Differential regulation of cell migration and proliferation through proline-rich tyrosine kinase 2 in endothelial cells

Proline-rich tyrosine kinase 2 (Pyk2), a member of the focal adhesion kinase family, is thought to act as a key component in vasculogenesis and angiogenesis. Therefore, we studied the effect of mutant Pyk2 expression on the migration and proliferation in endothelial cells (ECs). Two types of mutant Pyk2 were examined by adenovirus vectors AxCA-Pyk2K457A, expressing a kinase inactive mutant, and AxCA-Pyk2Y402F, expressing a tyrosine autophosphorylation site mutant, in addition to AxCA-Pyk2, expressing wild-type Pyk2. Migration of ECs infected with AxCA-Pyk2Y402F increased to a level similar to that of ECs infected with AxCA-Pyk2. The size of effect was dependent on the amount of applied adenoviruses within the range of 3-30 multiplicity of infection. In contrast, AxCA-Pyk2K457A infection did not show any significant effect on cell migration. Western blotting showed that both phosphorylation of Pyk2 Y(881) and association of p130(Cas) with Pyk2 were enhanced in ECs infected with AxCA-Pyk2Y402F as well as with AxCA-Pyk2, but not in ECs infected with AxCA-Pyk2K457A. Therefore, signaling mediated by Pyk2 Y(881) and p130(Cas) may be involved in the migration of ECs infected either with AxCA-Pyk2Y402F or with AxCA-Pyk2. In proliferation assay, AxCA-Pyk2 infection suppressed EC proliferation significantly; however, neither AxCA-Pyk2Y402F nor AxCA-Pyk2K457A showed such an inhibitory effect. Thus, the two Pyk2 mutants revealed that Pyk2 signaling differentially regulates cell migration and proliferation pathways.     

血管内皮生长因子增加内皮细胞通透性的基质金属蛋白酶-2机制

目的　研究血管内皮生长因子 (VEGF)增加血管内皮细胞对脂蛋白 (LDL)通透性与基质金属蛋白酶 2 (MMP 2 )的关系。方法　用异硫氰酸荧光素 (FITC)标记LDL ,荧光分光光度法测定FITC LDL的含量 ,在牛主动脉内皮细胞通透性模型上研究VEGF对FITC LDL通透率的影响 ;采用明胶酶谱法测定MMP 2活性。结果　VEGF剂量依赖性地增加血管内皮细胞对FITC LDL的通透性 ,MMP 2特异性抗体显著抑制VEGF的作用 ;VEGF还能剂量依赖性地升高MMP 2的活性。结论VEGF增加血管内皮细胞对FITC LDL的通透性的作用可能由MMP 2介导。     

Differential actions of PAR2 and PAR1 in stimulating human endothelial cell exocytosis and permeability: the role of Rho-GTPases

Endothelial cell proteinase activated receptors (PARs) belong to a family of heterotrimeric G protein-coupled receptors that are implicated in leukocyte accumulation and potentiation of reperfusion injury. We characterized the effect and the signal transduction pathways recruited after stimulation of endothelial PAR2. We used von Willebrand Factor (vWF) release and monolayer permeability to peroxidase to report Weibel-Palade body (WPB) exocytosis and pore formation, respectively. Human umbilical vein endothelial cells (HUVECs) were stimulated with the selective PAR2 agonist peptide SLIGRL-NH2 or PAR1 agonist peptide TFLLR-NH2. PAR2 stimulation resulted in WPB exocytosis like PAR1 stimulation but, unlike PAR1, failed to increase monolayer permeability. BAPTA-AM inhibited PAR2-induced exocytosis, indicating a PAR2 calcium-dependent signal in ECs. Moreover, PAR2-like PAR1-stimulated exocytosis requires actin cytoskeleton remodeling, because vWF release is inhibited if the cells were pretreated with Jasplakinolide. Rho-GTPase activity is required for PAR-stimulated exocytosis, because inactivation of this family of actin-regulatory proteins with Clostridium difficile toxin B blocked exocytosis. Expression of dominant-negative mutant Cdc42(17N) inhibited exocytosis whereas neither dominant-negative Rac(17N) expression nor C3 exotoxin treatment affected vWF release. PAR2 stimulated RhoA-GTP weakly compared with the PAR1 agonist. We conclude that both PAR2 and PAR1 elicit WP body exocytosis in a calcium and Cdc42 GTPase-dependent manner. In contrast, the differential effect of PAR1 versus PAR2 activation to increase monolayer permeability correlates with weak RhoA activation by the PAR2 agonist.     

Differential TIMP3 expression affects tumor progression and angiogenesis in melanomas through regulation of directionally persistent endothelial cell migration

The angiogenic potential of solid tumors, or the ability to initiate neovasculature development from pre-existing host vessels, is facilitated by soluble factors secreted by tumor cells and involves breaching of extracellular matrix barriers, endothelial cell (EC) proliferation, migration and reassembly. We evaluated the angiogenic potential of human melanoma cell lines differing in their degree of aggressiveness, based on their ability to regulate directionally persistent EC migration. We observed that conditioned-medium (CM) of the aggressive melanoma cell line BLM induced a high effective migratory response in ECs, while CMs of Mel57 and 1F6 had an inhibitory effect. Further, the melanoma cell lines exhibited a varied expression profile of tissue inhibitor of metalloproteinase-3 (TIMP3), detectable in the CM. TIMP3 expression inversely correlated with aggressiveness of the melanoma cell line, and ability of the respective CMs to induce directed EC migration. Interestingly, TIMP3 expression was found to be silenced in the BLM cell line, concurrent with its role as a tumor suppressor. Treatment with recombinant human TIMP3 and CM of modified, TIMP3 expressing, BLM cells mitigated directional EC migration, while CM of TIMP3 silenced 1F6 cells induced directed EC migration. The functional implication of TIMP3 expression on tumor growth and angiogenic potential in melanoma was evaluated in vivo. We observed that TIMP3 expression reduced tumor growth, angiogenesis and macrophage infiltration of BLM tumors while silencing TIMP3 increased tumor growth and angiogenesis of 1F6 tumors. Taken together, our results demonstrate that TIMP3 expression correlates with inhibition of directionally persistent EC migration and adversely affects the angiogenic potential and growth of melanomas.     

MARCKS protein mediates hydrogen peroxide regulation of endothelial permeability

Impairment of endothelial barrier function is implicated in many vascular and inflammatory disorders. One prevalent mechanism of endothelial dysfunction is an increase in reactive oxygen species under oxidative stress. Previous reports have demonstrated that hydrogen peroxide (H(2)O(2)), a highly stable reactive oxygen species that modulates physiological signaling pathways, also enhances endothelial permeability, but the mechanism of this effect is unknown. Here, we identify the actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) as a key mediator of the H(2)O(2)-induced permeability change in bovine aortic endothelial cells. MARCKS knockdown and H(2)O(2) treatment alter the architecture of the actin cytoskeleton in endothelial cells, and H(2)O(2) induces the phosphorylation and translocation of MARCKS from the cell membrane to the cytosol. Using pharmacological inhibitors and small interference RNA constructs directed against specific proteins, we uncover a signaling cascade from Rac1 to Abl1, phospholipase Cγ1, and PKCδ that is triggered by H(2)O(2) and leads to MARCKS phosphorylation. Our findings establish a distinct role for MARCKS in the regulation of H(2)O(2)-induced permeability change in endothelial cells, and suggest potential new therapeutic targets for the treatment of disorders involving oxidative stress and altered endothelial permeability.     

Role of endothelial cell cytoskeleton in control of endothelial permeability

Increased permeability of the pulmonary microvasculature is felt to cause acute noncardiogenic lung edema, and histological studies of edematous lungs show gaps between apparently healthy endothelial cells. To determine whether alterations in endothelial cell cytoskeletons would alter endothelial permeability, we exposed monolayers of pulmonary artery endothelial cells grown on micropore filters to cytochalasin B or D. Cytochalasin exposed monolayers demonstrated a 2- to 3-fold increase in endothelial permeability that was readily reversible by washing the monolayers free of cytochalasins. Parallel phase contrast and fluorescence microscopy demonstrated retraction of cell cytoplasm and disruption of bundles of microfilaments in cytochalasin exposed cells. These changes also were readily reversed after washing the cells free from cytochalasins. To test the relevance of these findings to an in situ microvasculature, we added cytochalasin B to the perfusate of isolated rabbit lungs and observed that cytochalasin B caused a high permeability lung edema. These studies suggest that endothelial cell cytoskeletons may be important determinants of endothelial permeability.     

Melatonin prevents endothelial cell pyroptosis via regulation of long noncoding RNA MEG3/miR‐223/NLRP3 axis

Atherosclerosis (AS ) is an inflammatory disease linked to endothelial dysfunction. Melatonin is reported to possess substantial anti‐inflammatory properties, which has proven to be effective in AS . Emerging literature suggests that pyroptosis plays a critical role during AS progression. However, whether pyroptosis contributes to endothelial dysfunction and the underlying molecular mechanisms remained unexploited. This study was designed to investigate the antipyroptotic effects of melatonin in atherosclerotic endothelium and to elucidate the potential mechanisms. In this study, high‐fat diet (HFD )‐treated ApoE^?/? mice were used as an atherosclerotic animal model. We found intragastric administration of melatonin for 12 weeks markedly reduced the atherosclerotic plaque in aorta. Meanwhile, melatonin also attenuated the expression of pyroptosis‐related genes, including NLRP 3, ASC , cleaved caspase1, NF ‐κB/GSDMD , GSDMD N‐termini, IL ‐1β, and IL ‐18 in aortic endothelium of melatonin‐treated animals. Consistent antipyroptotic effects were also observed in ox‐LDL ‐treated human aortic endothelial cells (HAEC s). We found that lncRNA MEG 3 enhanced pyroptosis in HAEC s. Moreover, MEG 3 acted as an endogenous sponge by sequence complementarity to suppress the function of miR‐223 and to increase NLRP 3 expression and enhance endothelial cell pyroptosis. Furthermore, knockdown of miR‐223 blocked the antipyroptotic actions of melatonin in ox‐LDL ‐treated HAEC s. Together, our results suggest that melatonin prevents endothelial cell pyroptosis via MEG 3/miR‐223/NLRP 3 axis in atherosclerosis, and therefore, melatonin replacement might be considered a new strategy for protecting endothelium against pyroptosis, thereby for the treatment of atherosclerosis associated with pyroptosis.     

cAMP and cGMP signaling cross-talk: role of phosphodiesterases and implications for cardiac pathophysiology

Cyclic nucleotide phosphodiesterases regulate cAMP-mediated signaling by controlling intracellular cAMP content. The cAMP-hydrolyzing activity of several families of cyclic nucleotide phosphodiesterases found in human heart is regulated by cGMP. In the case of PDE2, this regulation primarily involves the allosteric stimulation of cAMP hydrolysis by cGMP. For PDE3, cGMP acts as a competitive inhibitor of cAMP hydrolysis. Several cGMP-mediated responses in cardiac cells, including a potentiation of Ca(2+) currents and a diminution of the responsiveness to beta-adrenergic receptor agonists, have been shown to result from the effects of cGMP on cAMP hydrolysis. These effects appear to be dependent on the specific spatial distribution of the cGMP-generating and cAMP-hydrolyzing proteins, as well as on the intracellular concentrations of the two cyclic nucleotides. Gaining a more precise understanding of how these cross-talk mechanisms are individually regulated and coordinated is an important direction for future research.     

Cyclic GMP phosphodiesterases and regulation of smooth muscle function

Cyclic GMP (cGMP) made in response to atrial natriuretic peptide (ANP) or nitric oxide (NO) is an important regulator of short-term changes in smooth muscle tone and longer-term responses to chronic drug treatment or proliferative signals. The ability of smooth muscle cells (SMCs) to utilize different combinations of phosphodiesterase (PDE) isozymes allows cGMP to mediate these multiple processes. For example, PDE5 as a major cGMP-hydrolyzing PDE effectively controls the development of smooth muscle relaxation. In order for contraction to occur, PDE5 is activated and cGMP falls. Conversely, blockade of PDE5 activity allows the relaxation cycle to be prolonged and enhanced. A recently shown direct activation of PDE5 by cGMP binding to the GAF A domain suggests that this regulatory site might be a target for new drug development. The calcium surge associated with vasoconstrictor initiated contraction also activates a calcium/calmodulin-dependent PDE (PDE1A). Together, PDE5 and PDE1A lower cGMP sufficiently to allow contraction. Longer term, both PDE5 and PDE1A mRNA are induced by chronic stimulation of guanylyl cyclase. This induction is a major cause of the tolerance that develops to NO-releasing drugs. Finally, high levels of cGMP or cAMP also act as a brake to attenuate the proliferative response of SMCs to many mitogens. After vessel damage, in order for SMC proliferation to occur, the levels of cGMP and cAMP must be decreased. In humans, this decrease is caused in large part by induction of another Ca2+/calmodulin-dependent PDE (PDE1C) that allows the brake to be released and proliferation to start.     

Differential regulation of CXCR2 trafficking by Rab GTPases

Intracellular trafficking of chemokine receptors plays an important role in fine-tuning the functional responses of neutrophils and lymphocytes in the inflammatory process and HIV infection. Although many chemokine receptors internalize through clathrin-coated pits, regulation of the receptor trafficking is not fully understood. The present study demonstrated that CXCR2 was colocalized with transferrin and low-density lipoprotein (LDL) after agonist treatment for different periods of time, suggesting 2 intracellular trafficking pathways for this receptor. CXCR2 was colocalized with Rab5 and Rab11a, which are localized in early and recycling endosomes, respectively, in response to agonist stimulation for a short period of time, suggesting a recycling pathway for the receptor trafficking. However, overexpression of a dominant-negative Rab5-S34N mutant significantly attenuated CXCR2 sequestration. The internalized CXCR2 was recycled back to the cell surface after removal of the agonist and recovery of the cells, but receptor recycling was inhibited by overexpression of a dominant-negative Rab11a-S25N mutant. After prolonged (4-hour) agonist treatment, CXCR2 exhibited significantly increased colocalization with Rab7, which is localized in late endosomes. The colocalization of CXCR2 with LDL and LAMP-1 suggests that CXCR2 is targeted to lysosomes for degradation after prolonged ligand treatment. However, the colocalization of CXCR2 with Lamp1 was blocked by the overexpression of a dominant-negative Rab7-T22N mutant. In cells overexpressing Rab7-T22N, CXCR2 was retained in the Rab5- and Rab11a-positive endosomes after prolonged (4-hour) agonist treatment. Our data suggest that the intracellular trafficking of CXCR2 is differentially regulated by Rab proteins.     

Extracellular RNA mediates endothelial-cell permeability via vascular endothelial growth factor

Cell injury leads to exposure of intracellular material and is associated with increased permeability of vessels in the vicinity of the damage. Here, we demonstrate that natural extracellular RNA as well as artificial RNA (poly-I:C), or single-stranded RNA but not DNA, significantly increased the permeability across brain microvascular endothelial cells in vitro and in vivo. RNA-induced hyperpermeability of tight monolayers of endothelial cells correlated with disintegration of tight junctions and was mediated through vascular endothelial growth factor (VEGF), reminiscent of heparin's activities. Antisense oligonucleotides against VEGF-receptor 2 (VEGF-R2) prevented the permeability-inducing activity of extracellular RNA and heparin completely. Hence, these polyanionic substances can lead to mobilization/stabilization of VEGF with the subsequent activation of VEGF-R2. In accordance with these functional data, strong binding of VEGF as well as other growth factors to RNA was demonstrable. In in vivo rat models of FeCl(3)-induced sinus sagittal is superior thrombosis and stroke/brain edema, pretreatment of animals with RNase (but not DNase) resulted in a significant reduction of vessel occlusion, infarct volume, and prevention of brain edema formation. Together, these results identify extracellular RNA as a novel natural permeability factor, upstream of VEGF, whereas counteracting RNase treatment may serve as new vessel-protective modality.     

Acute temporal regulation of vascular endothelial growth/permeability factor expression and endothelial morphology in the baboon endometrium by ovarian steroids

We recently showed that endometrial vascular endothelial growth/permeability factor (VEG/PF) mRNA expression was decreased by ovariectomy of baboons and restored by chronic administration of estrogen. However, it remains to be determined whether this effect of estrogen reflects genomic up-regulation of VEG/PF and leads to an increase in microvascular permeability, an early physiological event in angiogenesis. Therefore, we determined the temporal expression of VEG/PF mRNA in glandular epithelial and stromal cells isolated by laser capture microdissection from and width of microvascular paracellular clefts that regulate vessel permeability in the endometrium of ovariectomized baboons after acute estradiol and/or progesterone administration. Endometrial VEG/PF mRNA levels were increased in five of five animals within 2 h of estradiol administration and remained elevated at 4 and 6 h. The net increase in glandular epithelial (7.31 +/- 2.72 attomol/fmol 18S ribosomal rRNA) and stromal (3.13 +/- 0.36) cell VEG/PF mRNA levels after estradiol administration was over 8-fold (P < 0.05) and 2.6-fold (P < 0.01) greater, respectively, than after vehicle (0.90 +/- 0.30, glands and 1.20 +/- 0.33, stroma). In contrast, endometrial VEG/PF mRNA expression was unaltered by progesterone. After estradiol treatment, endometrial paracellular cleft width was increased (P < 0.01) from a mean (+/-SE) of 71.6 +/- 4.6 nm at 0 h to 101.1 +/- 6.4 nm at 6 h, whereas vehicle or progesterone had no effect. We suggest that estrogen has a major role in regulating VEG/PF synthesis and early events in angiogenesis in the primate endometrium.     

Differential regulation of rodent hepatocyte and oval cell proliferation by interferon gamma

Hepatocytes and intrahepatic progenitor cells (oval cells) have similar responses to most growth factors but rarely proliferate together. Oval cells constitute a reserve compartment that is activated when hepatocyte proliferation is inhibited. Interferon gamma (IFN-gamma) increases in liver injury that involves oval cell responses, but it is not upregulated during liver regeneration after partial hepatectomy. Based on these observations, we used well-characterized lines of hepatocytes (AML-12 cells) and oval cells (LE-6 cells) to investigate the potential mechanisms that regulate differential growth responses in hepatocytes and oval cells. We show that IFN-gamma blocks hepatocyte proliferation in vivo, and that in combination with either tumor necrosis factor (TNF) or lipopolysaccharide (LPS), it causes cell cycle arrest in hepatocytes but stimulates oval cell proliferation in cultured cells. The hepatocyte cell cycle arrest is reversible, is p53-independent, and is not associated with apoptosis. Treatment of AML-12 hepatocytes with IFN-gamma/LPS or IFN-gamma/TNF, but not with individual cytokines, induced NO synthase and generated NO, while similarly treated oval cells produced little if any NO. Generation of NO by an NO donor reproduced the inhibitory effect of the cytokine combinations on AML-12 cell replication, while NO inhibitors abolish the replication deficiency. In conclusion, we propose that IFN-gamma, in conjunction with TNF or LPS, can both inhibit hepatocyte proliferation through the generation of NO and stimulate oval cell replication. The response of hepatocytes and oval cells to cytokine combinations may contribute to the differential proliferation of these cells in hepatic growth processes.