The C-terminal 26-residue peptide of serpin A1 is an inhibitor of HIV-1

The signal transduction pathway involved in hepatocyte growth factor (HGF)-induced capillary morphogenesis of endothelial cells was investigated. HGF-induced capillary morphogenesis of the murine spleen endothelial cell line MSS31 was inhibited by a Src family kinase inhibitor, PP2. Stable expression of kinase-inactive Src in MSS31 cells inhibited HGF-induced activation of Src as well as capillary morphogenesis. The HGF-induced capillary morphogenesis of human umbilical vein endothelial cells was also inhibited by PP2 and was reduced by the downregulation of Src by small interfering RNA. These results suggest that HGF induces capillary morphogenesis of endothelial cells through Src.     

Src family kinases regulate p38 MAPK-mediated IL-6 production in Kupffer cells following hypoxia

Tissue hypoxia is a common sequel of trauma-hemorrhage but can occur even without blood loss under hypoxic conditions. Although hypoxia is known to upregulate Kupffer cells (KC) to release cytokines, the precise mechanism of release remains unknown. We hypothesized that Src family kinases play a role in mediating KC mitogen-activated protein kinase (MAPK) signaling and their cytokine production after hypoxia. Male C3H/HeN mice received either Src inhibitor PP1 (1.5 mg/kg body wt) or vehicle 1 h before hypoxia. KCs were isolated 1 h after hypoxia, lysed, and immunoblotted with antibodies to Src, p38, ERK1/2, or JNK proteins. In addition, KCs were cultured to measure interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production. Hypoxia produced a significant increase in KC Src and MAPK (p38, ERK, JNK) activity compared with normoxic controls. This was associated with an increase in IL-6 and MCP-1 production. Treatment with PP1 abolished the increase in KC Src activation as well as p38 activity. However, PP1 did not prevent the increase in KC ERK1/2 or JNK phosphorylation. Furthermore, administration of PP1 prevented the hypoxia-induced increase in IL-6 but not MCP-1 release by KC. Additional in vitro results suggest that p38 but not ERK1/2 or JNK are critical for KC IL-6 production. In contrast, the production of MCP-1 by KC was found to be independent of MAPK. Thus hypoxia increases KC IL-6 production by p38 MAPK activation via Src-dependent pathway. Src kinases may therefore be a novel therapeutic target for preventing immune dysfunction following low-flow conditions in trauma patients. innate immunity; macrophages; cell signaling     

Src and Pyk2 mediate angiotensin II effects in cultured rat astrocytes

Angiotensin II (Ang II)-induced proliferation of rat astrocytes is mediated by multiple signaling pathways. In the present study, we investigated the role of non-receptor tyrosine kinases on Ang II-signaling and proliferation of astrocytes cultured from neonatal rat pups. Ang II stimulated astrocyte growth, ERK1/2 phosphorylation and the phosphorylation of Src and proline-rich tyrosine kinase-2 (Pyk2), in astrocytes obtained from brainstem and cerebellum. Pretreatment with 10 microM PP2, a selective Src inhibitor, inhibited Ang II stimulated ERK1/2 phosphorylation by 59% to 91% both in brainstem and cerebellum astrocytes. PP2 also inhibited Ang II induction of brainstem (76% inhibition) and cerebellar (64% inhibition) astrocyte growth. Similarly, pretreatment with 25 microM dantrolene, the Pyk2 inhibitor, attenuated ERK1/2 activity in brainstem (62% inhibition) and in cerebellum astrocytes (44% inhibition). Interestingly, inhibition of Pyk2 inhibited Ang II-induced Src activation suggesting that these two non-receptor tyrosine kinases may be acting in concert to mediate Ang II effects in astrocytes. In summary, we found that Ang II stimulates the non-receptor tyrosine kinases Src and Pyk2 which mediate Ang II-induced ERK1/2 activation leading to stimulation of astrocyte growth. In addition, these two tyrosine kinases may be interacting to regulate effects of the peptide in these cells.     

Fibroblast growth factor-2-mediated capillary morphogenesis of endothelial cells requires signals via Flt-1/vascular endothelial growth factor receptor-1: possible involvement of c-Akt

Capillary morphogenesis is a crucial angiogenic response of endothelial cells. Although fibroblast growth factor-2 (FGF-2) potently induces capillary morphogenesis, the contribution of vascular endothelial growth factor-A (VEGF-A) in this response has not been clarified well. Here we examined the role of VEGF signaling in FGF-2-induced capillary morphogenesis by murine brain capillary endothelial cells (IBE cells) and human umbilical vein endothelial cells. FGF-2-treated IBE cells rapidly extended on Matrigel in association with actin reorganization. Chimeric protein, of which the extracellular domain of VEGF receptor-1 (VEGFR-1) fused to immunoglobulin Fc, inhibited FGF-2-induced cell extension, resulting in decreased capillary morphogenesis. Blocking antibody against VEGFR-1 inhibited FGF-2-induced capillary formation. Also, anti-VEGF-A antibody inhibited FGF-2-induced capillary morphogenesis, which was restored by the addition of placental growth factor-1. Similar results were obtained by the experiments with human umbilical vein endothelial cells. Expression of kinase-inactive c-Akt in IBE cells showed impaired capillary morphogenesis promoted by FGF-2. Conversely, stable cell lines expressing activated c-Akt demonstrated ligand-independent capillaries, which were resistant to the treatment with anti-VEGFR-1 blocking antibody. Upstream of c-Akt, calmodulin-dependent signals seemed to be involved. Taken together, signals via VEGFR-1 were required for FGF-2-induced capillary morphogenesis by endothelial cells, and c-Akt activity seemed to be involved in this process.     

Hypoxia induces differential expression of the integrin receptors alpha(vbeta3) and alpha(vbeta5) in cultured human endothelial cells

The integrins alpha(vbeta3) and alpha(vbeta5) have been implicated in playing a key role in the process of angiogenesis. In this study, we examined the effects of hypoxia, an important stimulus of angiogenesis, on the differential expression of the integrin subunits beta(3) and beta(5). beta(3) and beta(5) messenger RNA (mRNA), protein levels, and alpha(v)beta(3) function were measured in human umbilical vein endothelial cells (HUVECs) cultured under normoxic and hypoxic (1% O(2)) conditions. Cells exposed to hypoxic conditions for up to 72 h showed gradually increased mRNA levels of alpha(V) and beta(3), peaking at 24 h, in comparison with cells cultured under normoxic conditions. However, beta(5) mRNA levels, under the same hypoxic conditions, remained at a constant level. Results from Western blot analysis of HUVECs, cultured under hypoxic conditions, paralleled those of the Northern analysis with an increased expression in alpha(v)beta(3) protein levels, measured by blotting with LM609, evident by 24 h. alpha(v)beta(5) protein levels, measured by blotting with P1F6, did not change for up to 72 h. HUVECs cultured under hypoxic conditions for 72 h showed increased attachment to fibrinogen, an alpha(v)beta(3) mediated process. These results indicate that hypoxia can increase expression of alpha(v)beta(3) in HUVECs, and that hypoxic regulation of alpha(v)beta(3) may be an important regulator of angiogenesis.     

Angiotensin II induces epithelial-to-mesenchymal transition in renal epithelial cells through reactive oxygen species/Src/caveolin-mediated activation of an epidermal growth factor receptor-extracellular signal-regulated kinase signaling pathway

Chronic activation of the renin-angiotensin system plays a deleterious role in progressive kidney damage, and the renal proximal tubule is known to play an important role in tubulointerstitial fibrosis; however, the underlying molecular mechanism is unclear. Here we report that in the proximal tubule-like LLCPKcl4 cells expressing angiotensin II (Ang II) type 1 receptor, Ang II induced changes in cell morphology and expression of epithelial-to-mesenchymal transition (EMT) markers, which were inhibited by the miotogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK)-activating kinase (MEK) inhibitor PD98059 or the Src kinase inhibitor PP2. Ang II-stimulated phosphorylation of caveolin-1 (Cav) at Y14 and epidermal growth factor receptor (EGFR) at Y845 and induced association of these phosphoproteins in caveolin-enriched lipid rafts, thereby leading to prolonged EGFR-ERK signaling that was inhibited by Nox4 small interfering RNA (siRNA) and Src siRNA. Two different antioxidants not only inhibited phosphorylation of Src at Y416 but also blocked the EGFR-ERK signaling. Moreover, erlotinib (the EGFR tyrosine kinase inhibitor), EGFR siRNA, and Cav siRNA all inhibited both prolonged EGFR-ERK signaling and phenotypic changes induced by Ang II. Thus, this report provides the first evidence that reactive oxygen species (ROS)/Src-dependent activation of persistent Cav-EGFR-ERK signaling mediates renal tubular cell dedifferentiation and identifies a novel molecular mechanism that may be involved in progressive renal injury caused by chronic exposure to Ang II.     

Src tyrosine kinase inhibitor PP2 suppresses ERK1/2 activation and epidermal growth factor receptor transactivation by X-irradiation

Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. Specific epidermal growth factor receptor (EGFR) inhibitor AG1478 and specific Src inhibitor PP2 inhibited IR-induced ERK1/2 activation but phosphatidylinositol-3 kinase inhibitor wortmannin did not. IR caused EGFR tyrosine phosphorylation, whereas it did not induce EGFR autophosphorylation at Tyr992, Tyr1045, and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. SHP-2, which positively regulates EGFR/Ras/ERK signaling cascade, became activated by IR as indicated by its phosphorylation at Tyr542. This activation was inhibited by PP2 not by AG1478, which suggests Src-dependent activation of SHP-2. Src and PTPalpha, which positively regulates Src, became activated as indicated by phosphorylation at Tyr416 and Tyr789, respectively. These data suggest that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation.     

Exosomes Derived from Hypoxic Leukemia Cells Enhance Tube Formation in Endothelial Cells

Hypoxia plays an important role during the evolution of cancer cells and their microenvironment. Emerging evidence suggests communication between cancer cells and their microenvironment occurs via exosomes. This study aimed to clarify whether hypoxia affects angiogenic function through exosomes secreted from leukemia cells. We used the human leukemia cell line K562 for exosome-generating cells and human umbilical vein endothelial cells (HUVECs) for exosome target cells. Exosomes derived from K562 cells cultured under normoxic (20%) or hypoxic (1%) conditions for 24 h were isolated and quantitated by nanoparticle tracking analysis. These exosomes were then cocultured with HUVECs to evaluate angiogenic activity. The exosomes secreted from K562 cells in hypoxic conditions significantly enhanced tube formation by HUVECs compared with exosomes produced in normoxic conditions. Using a TaqMan low-density miRNA array, we found a subset of miRNAs, including miR-210, were significantly increased in exosomes secreted from hypoxic K562 cells. We demonstrated that cancer cells and their exosomes have altered miRNA profiles under hypoxic conditions. Although exosomes contain various molecular constituents such as proteins and mRNAs, altered exosomal compartments under hypoxic conditions, including miR-210, affected the behavior of endothelial cells. Our results suggest that exosomal miRNA derived from cancer cells under hypoxic conditions may partly affect angiogenic activity in endothelial cells.     

缺氧时肺动脉内皮细胞与肺动脉平滑肌细胞增殖的相互影响

血管内皮细胞和血管平滑细胞在结构和功能上关系密切,两者的相互在与血管舒缩笔血和壁结构。本文观察了培养的小牛肺动脉内皮细胞（ＰＡＥＣｓ）和肺动平滑肌细胞（ＰＡＳＭＣＳ）缺氧时在细胞增殖方面的相互影响。ＰＡＳＭＣＳ常氧条件培养基（ＣＭ）可使ＰＡＥＣＳ的＾３Ｈ－ＴｄＲ掺入降低约５８％,缺氧ＣＭ对ＰＡＥＣＳ的＾３Ｈ－ＴｄＲ掺入无明显的抑制作用;ＰＡＥＣＳ的常氧ＣＭ使ＰＡＳＭＳ的＾３Ｈ－ＴｄＲ掺入升高约６０     

TNF-related activation-induced cytokine (TRANCE) induces angiogenesis through the activation of Src and phospholipase C (PLC) in human endothelial cells.

Angiogenesis is an essential step for many physiological and pathological processes. Tumor necrosis factor (TNF) superfamily cytokines are increasingly recognized as key modulators of angiogenesis. In this study, we tested whether TNF-related activation-induced cytokine (TRANCE), a new member of the TNF superfamily, possesses angiogenic activity in vitro and in vivo. TRANCE stimulated DNA synthesis, chemotactic motility, and capillary-like tube formation in primary cultured human umbilical vein endothelial cells (HUVECs). Both Matrigel plug assay in mice and chick chorioallantoic membrane assay revealed that TRANCE potently induced neovascularization in vivo. TRANCE had no effect on vascular endothelial growth factor (VEGF) expression in HUVECs and TRANCE-induced angiogenic activity was not suppressed by VEGF-neutralizing antibody, implying that TRANCE-induced angiogenesis may be the result of its direct action on endothelial cells. TRANCE evoked a time- and dose-dependent activation of the mitogen-activated protein kinases ERK1/2 and focal adhesion kinase p125(FAK) in HUVECs, which are closely linked to angiogenesis. These signaling events were blocked by the Src inhibitor PP1 or the phospholipase C (PLC) inhibitor. Furthermore, these inhibitors and the Ca(2+) chelator BAPTA-AM suppressed TRANCE-induced HUVEC migration. These results indicate that the angiogenic activity of TRANCE is mediated through the Src-PLC-Ca(2+) signaling cascade upon receptor engagement in endothelial cells, suggesting the role of TRANCE in neovessel formation under physiological and pathological conditions.     

Activation of adenovirus type 2 early region 4 ORF4 cytoplasmic death function by direct binding to Src kinase domain

Adenovirus type 2 (Ad2) early region 4 ORF4 (E4orf4) triggers a major death pathway that requires its accumulation in cellular membranes and its tyrosine phosphorylation. This program is regulated by Src family kinases and triggers a potent ZVAD (benzyloxycarbonyl-VAD)- and Bcl2-resistant cell death response in human-transformed cells. How E4orf4 deregulates Src-dependent signaling is unknown. Here we provide strong evidence that a physical interaction requiring the kinase domain of Src and the arginine-rich motif of E4orf4 is involved. The Src binding domain of E4orf4 overlaps with, but is distinct from that of the Balpha subunit of protein phosphatase 2A (PP2A-Balpha) and some E4orf4 complexes contain both PP2A and Src. Functional assays using mutant E4orf4 revealed that deregulation of Src signaling, activation of the Jun kinase pathway, and cell blebbing were all critically dependent on Src binding. In contrast, PP2A-Balpha binding per se was not required to engage the Src-dependent death pathway but was more critical for triggering a distinct death activity. Both E4orf4 death activities were manifested within a given cell population, were typified by distinct morphological features, and contributed to overall cell killing, although to different extents in various cell types. We conclude that E4orf4 binding to the Src kinase domain leads to deregulation of Src signaling and plays a crucial role in induction of the cytoplasmic death pathway. Nonetheless, both Src and PP2A enzymes are critical targets of E4orf4 that likely cooperate to trigger E4orf4-induced tumor cell killing and whose relative contributions may vary in function of the cellular background.     

Downregulation of Fes inhibits VEGF-A-induced chemotaxis and capillary-like morphogenesis by cultured endothelial cells

The aim of this study was to determine whether the downregulation of endogenous Fes by siRNA in cultured endothelial cells affects vascular endothelial growth factor-A (VEGF-A)-induced chemotaxis and capillary-like morphogenesis, which are considered as angiogenic cellular responses in vitro. VEGF-A-treatment induced autophosphorylation of Fes in cultured endothelial cells. LY294002, a phosphoinositide 3-kinase inhibitor, significantly inhibited VEGF-A-induced chemotaxis and capillary-like morphogenesis. Downregulation of Fes attenuated these VEGF-A-induced cellular responses but LY294002 did not produce further inhibition of these responses. Downregulation of Fes neither affected VEGF-A-induced autophosphorylation of VEGF receptor 2 nor mitogen-activated protein kinase activation, but markedly decreased Akt activation. Taken together, our novel results indicate the involvement of Fes in VEGF-A-induced cellular responses by cultured endothelial cells.     

VEGFR2 and Src kinase inhibitors suppress Andes virus-induced endothelial cell permeability

Hantaviruses predominantly infect human endothelial cells and, in the absence of cell lysis, cause two diseases resulting from increased vascular permeability. Andes virus (ANDV) causes a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). ANDV infection enhances the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF) by increasing signaling responses directed by the VEGFR2-Src-VE-cadherin pathway, which directs adherens junction (AJ) disassembly. Here we demonstrate that inhibiting pathway-specific VEGFR2 and Src family kinases (SFKs) blocks ANDV-induced endothelial cell permeability. Small interfering RNA (siRNA) knockdown of Src within ANDV-infected endothelial cells resulted in an ～70% decrease in endothelial cell permeability compared to that for siRNA controls. This finding suggested that existing FDA-approved small-molecule kinase inhibitors might similarly block ANDV-induced permeability. The VEGFR2 kinase inhibitor pazopanib as well as SFK inhibitors dasatinib, PP1, bosutinib, and Src inhibitor 1 dramatically inhibited ANDV-induced endothelial cell permeability. Consistent with their kinase-inhibitory concentrations, dasatinib, PP1, and pazopanib inhibited ANDV-induced permeability at 1, 10, and 100 nanomolar 50% inhibitory concentrations (IC(50)s), respectively. We further demonstrated that dasatinib and pazopanib blocked VE-cadherin dissociation from the AJs of ANDV-infected endothelial cells by >90%. These findings indicate that VEGFR2 and Src kinases are potential targets for therapeutically reducing ANDV-induced endothelial cell permeability and, as a result, capillary permeability during HPS. Since the functions of VEGFR2 and SFK inhibitors are already well defined and FDA approved for clinical use, these findings rationalize their therapeutic evaluation for efficacy in reducing HPS disease. Endothelial cell barrier functions are disrupted by a number of viruses that cause hemorrhagic, edematous, or neurologic disease, and as a result, our findings suggest that VEGFR2 and SFK inhibitors should be considered for regulating endothelial cell barrier functions altered by additional viral pathogens.     

c-Jun N-terminal kinase activation by hydrogen peroxide in endothelial cells involves SRC-dependent epidermal growth factor receptor transactivation

The phenotypic properties of the endothelium are subject to modulation by oxidative stress, and the c-Jun N-terminal kinase (JNK) pathway is important in mediating cellular responses to stress, although activation of this pathway in endothelial cells has not been fully characterized. Therefore, we exposed endothelial cells to hydrogen peroxide (H(2)O(2)) and observed rapid activation of JNK within 15 min that involved phosphorylation of JNK and c-Jun and induction of AP-1 DNA binding activity. Inhibition of protein kinase C and phosphoinositide 3-kinase did not effect JNK activation. In contrast, the tyrosine kinase inhibitors, genistein, herbimycin A, and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) significantly attenuated H(2)O(2)-induced JNK activation as did endothelial cell adenoviral transfection with a dominant-negative form of Src, implicating Src as an upstream activator of JNK. Activation of JNK by H(2)O(2) was also inhibited by AG1478 and antisense oligonucleotides directed against the epidermal growth factor receptor (EGFR), implicating the EGFR in this process. Consistent with this observation, H(2)O(2) stimulated EGFR tyrosine phosphorylation and complex formation with Shc-Grb2 that was abolished by PP2, implicating Src in H(2)O(2)-induced EGFR activation. Tyrosine phosphorylation of the EGFR by H(2)O(2) did not involve receptor autophosphorylation at Tyr(1173) as assessed by an autophosphorylation-specific antibody. These data indicate that H(2)O(2)-induced JNK activation in endothelial cells involves the EGFR through an Src-dependent pathway that is distinct from EGFR ligand activation. These data represent one potential pathway for mediating oxidative stress-induced phenotypic changes in the endothelium.     

Angiopoietin-1 prevents VEGF-induced endothelial permeability by sequestering Src through mDia

Vascular endothelial growth factor (VEGF) and Angiopoietin 1 (Ang1) are both potent proangiogenic factors, but, whereas VEGF causes vascular permeability, Ang1 stabilizes blood vessels and protects them from VEGF-induced plasma leakage. The antivascular permeability mechanisms deployed by Ang1 are still undefined. Here, we demonstrate that Ang1 halts the ability of VEGF to induce the phosphorylation-dependent redistribution of the adhesion molecule VE-cadherin, thereby rescuing the endothelial barrier function. Ang1 inhibits the activation of Src by VEGF, the most upstream component of the pathway linking VEGF receptors to VE-cadherin internalization. Indeed, Ang1 promotes the activation of mDia through RhoA, resulting in the association of mDia with Src. This ultimately deprives VEGF receptors of an essential molecule required for promoting the disruption of endothelial cell-cell contacts and paracellular permeability.     

Inhibition of Src by direct interaction with protein phosphatase 2A

In this study, we report that Src kinase is inhibited by protein phosphatase 2A (PP2A), a serine/threonine phosphatase. We carried out experiments in vitro using purified PP2A (AC dimer) and full-length v-Src or truncated forms of v-Src. The inhibition of v-Src by PP2A is concentration- and time-dependent. Addition of okadaic acid, a PP2A phosphatase inhibitor, abolished the PP2A-dependent inhibition of v-Src. When experiments were carried out at 4 degrees C under conditions where PP2A activity is inhibited, Src activity was unaffected by the presence of PP2A, suggesting that PP2A binding alone is insufficient to block Src activity. These results imply that PP2A activity is essential for inhibition of v-Src. We also demonstrate that PP2A binds to the catalytic and the regulatory domains of v-Src.     

Effects of a dammarane-type saponin,ginsenoside Rd,in nicotine-induced vascular endothelial injury

BackgroundPanax notoginseng (Burk.) F.H. Chen is a traditional medicinal plant widely used to prevent and treat cardiovascular diseases. Ginsenoside Rd (GRd) is a major bioactive component of P. notoginseng, but specific effects on cardiovascular disease-related pathogenic processes are rarely studied, especially vascular endothelial injury.PurposeThis study investigated the potential protective efficacy of GRd against nicotine-induced vascular endothelial cell injury, disruption of vascular nitric oxide (NO) signaling, aberrant endothelium–monocyte adhesion, platelet aggregation, and vasoconstriction.Study design/MethodsVascular endothelial injury and functional disruption were investigated in cultured human umbilical vein endothelial cells (HUVECs) by biochemical assays for nitric oxide (NO) and angiotensin II (Ang II), immunofluorescence (IF) and western blotting for expression analyses of apoptosis- related proteins, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), Ang II type receptor 1 (AGTR1), toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappa B (NF-κB). In addition, vascular protection by GRd was examined in nicotine-administered Sprague-Dawley (SD) rats by serum NO and Ang II assays, and by hematoxylin-eosin (HE) and immunostaining of aorta. We also examined effects of GRd on monocyte (THP-1 cells) adhesion assays, adenosine diphosphate (ADP)-induced platelet aggregation, and phenylephrine (PE)-induced vasoconstriction of isolated rat aortic rings.ResultsIn HUVECs, nicotine significantly suppressed NO production, enhanced Ang II production, downregulated eNOS expression, and upregulated expression levels of AGTR1, TLR4, MyD88, NF-κB, iNOS, Bax/Bcl-2 ratio, cleaved caspase-3, and cytochrome c (cyt c). All of these changes were significantly reversed by GRd. In rats, oral GRd reversed the reduction NO and enhanced Ang II production in serum induced by nicotine administration, and HE staining revealed protection of aortic endothelial cells. In addition, GRd reversed nicotine-mediated enhancement of HUVECs-monocyte adhesion, inhibited ADP-induced platelet aggregation and PE-induced vasoconstriction.ConclusionGRd may prevent nicotine-induced cardiovascular diseases by preserving normal vascular endothelial NO signaling, suppressing platelet aggregation and vasoconstriction, and by preventing endothelial cell–monocyte adhesion.