Thrombolytic treatment of ischemic stroke with tissue plasminogen activator (tPA) is markedly limited owing to concerns about hemorrhagic complications and the requirement that tPA be administered ...within 3 h of symptoms. Here we report that tPA activation of latent platelet-derived growth factor-CC (PDGF-CC) may explain these limitations. Intraventricular injection of tPA or active PDGF-CC, in the absence of ischemia, leads to significant increases in cerebrovascular permeability. In contrast, co-injection of neutralizing antibodies to PDGF-CC with tPA blocks this increased permeability, indicating that PDGF-CC is a downstream substrate of tPA within the neurovascular unit. These effects are mediated through activation of PDGF-alpha receptors (PDGFR-alpha) on perivascular astrocytes, and treatment of mice with the PDGFR-alpha antagonist imatinib after ischemic stroke reduces both cerebrovascular permeability and hemorrhagic complications associated with late administration of thrombolytic tPA. These data demonstrate that PDGF signaling regulates blood-brain barrier permeability and suggest potential new strategies for stroke treatment.
Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of tissue-type and urokinase-type plasminogen activators (tPA and uPA, respectively). PAI-1 also interacts with non-proteinase ...targets such as vitronectin, heparin, and several endocytic receptors of the low-density lipoprotein-receptor family, including the low-density lipoprotein-receptor related protein (LRP) and the very low-density lipoprotein receptor (VLDLr). PAI-1 is a multifunctional protein that is not only a physiologic regulator of fibrinolysis and cell migration but is also associated with several acute and chronic pathologic conditions. PAI-1 is involved in the pathophysiology of renal, pulmonary, cardiovascular, and metabolic diseases, and in vitro experiments and animal studies have elucidated PAI-1's contribution to the physiology or pathology of some of these conditions. PAI-1 is normally present at low levels in plasma, but acute and chronic diseases are strongly associated with increased PAI-1 expression and release. At sites of vascular injury and inflammation, local PAI-1 levels are even higher, due to its concentration in extracellular matrix through association with vitronectin. Elevated local or systemic PAI-1 is not only a marker of disease; it can also exacerbate pathologic conditions. Thus, interventions that directly target PAI-1 may be useful for the treatment of a number of chronic and acute disorders. Typically, such interventional strategies would involve the identification of small molecule inhibitors of PAI-1, and several recent reviews have covered this topic. However, it may also be possible or even potentially advantageous, to exploit the diverse functional interactions of PAI-1 to create highly specific and targeted therapeutic agents based on the PAI-1 protein itself. To understand how PAI-1 could be developed as a therapeutic agent, it is first necessary to discuss its structural and functional characteristics in depth.
The inactivation of plasminogen activator inhibitor-1 (PAI-1) by the small molecule PAI-1 inhibitor PAI-039 (tiplaxtinin) has been investigated using enzymatic analysis, direct binding studies, ...site-directed mutagenesis, and molecular modeling studies. Previously PAI-039 has been shown to exhibit in vivo activity in various animal models, but the mechanism of inhibition is unknown. PAI-039 bound specifically to the active conformation of PAI-1 and exhibited reversible inactivation of PAI-1 in vitro. SDS-PAGE indicated that PAI-039 inactivated PAI-1 predominantly through induction of PAI-1 substrate behavior. Preincubation of PAI-1 with vitronectin, but not bovine serum albumin, blocked PAI-039 activity while analysis of the reciprocal experiment demonstrated that preincubation of PAI-1 with PAI-039 blocked the binding of PAI-1 to vitronectin. Together, these data suggest that the site of interaction of the drug on PAI-1 is inaccessible when PAI-1 is bound to vitronectin and may overlap with the PAI-1 vitronectin binding domain. This was confirmed by site-directed mutagenesis and molecular modeling studies, which suggest that the binding epitope for PAI-039 is localized adjacent to the previously identified interaction site for vitronectin. Thus, these studies provide a detailed characterization of the mechanism of inhibition of PAI-1 by PAI-039 against free, but not vitronectin-bound PAI-1, suggesting for the first time a novel pool of PAI-1 exists that is vulnerable to inhibition by inactivators that bind at the vitronectin binding site.
Plasminogen activator inhibitor type 1, (PAI-1) the primary inhibitor of the tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, has been implicated in a wide range of pathological ...processes, making it an attractive target for pharmacologic inhibition. Currently available small-molecule inhibitors of PAI-1 bind with relatively low affinity and do not inactivate PAI-1 in the presence of its cofactor, vitronectin. To search for novel PAI-1 inhibitors with improved potencies and new mechanisms of action, we screened a library selected to provide a range of biological activities and structural diversity. Five potential PAI-1 inhibitors were identified, and all were polyphenolic compounds including two related, naturally occurring plant polyphenols that were structurally similar to compounds previously shown to provide cardiovascular benefit in vivo. Unique second generation compounds were synthesized and characterized, and several showed IC50 values for PAI-1 between 10 and 200 nm. This represents an enhanced potency of 10–1000-fold over previously reported PAI-1 inactivators. Inhibition of PAI-1 by these compounds was reversible, and their primary mechanism of action was to block the initial association of PAI-1 with a protease. Consistent with this mechanism and in contrast to previously described PAI-1 inactivators, these compounds inactivate PAI-1 in the presence of vitronectin. Two of the compounds showed efficacy in ex vivo plasma and one blocked PAI-1 activity in vivo in mice. These data describe a novel family of high affinity PAI-1-inactivating compounds with improved characteristics and in vivo efficacy, and suggest that the known cardiovascular benefits of dietary polyphenols may derive in part from their inactivation of PAI-1.
eNOS (endothelial nitric oxide synthase) activity is post-translationally regulated in a complex fashion by acylation, protein-protein interactions, intracellular trafficking and phosphorylation, ...among others. Signalling pathways that regulate eNOS activity include phosphoinositide 3-kinase/Akt, cyclic nucleotide-dependent kinases PKA (protein kinase A) and PKG, PKC, as well as ERKs (extracellular-signal-regulated kinases). The role of ERKs in eNOS activation remains controversial. In the present study, we have examined the role of ERK1/2 in eNOS activation in HUVEC-CS transformed HUVEC (human umbilical-vein endothelial cells) as well as a widely used model for eNOS study, transiently transfected COS-7 cells. U0126 pretreatment of HUVEC-CS potentiated ATP-stimulated eNOS activity, independent of changes in intracellular Ca2+ concentration (Ca2+i). In COS-7 cells transiently expressing ovine eNOS, U0126 potentiated A23187-stimulated eNOS activity, but inhibited ATP-stimulated activity. Compensatory changes in phosphorylation of five key eNOS residues did not account for changes in A23187-stimulated activity. However, in the case of ATP, altered phosphorylation and changes in Ca2+i may partially contribute to U0126 inhibition of activity. Finally, seven eNOS alanine mutants of putative ERK1/2 targets were generated and the effects of U0126 pretreatment on eNOS activity were gauged with A23187 and ATP treatment. T97A-eNOS was the only construct significantly different from wild-type after U0126 pretreatment and ATP stimulation of eNOS activation. In the present study, eNOS activity was either potentiated or inhibited in COS-7 cells, suggesting agonist dependence for MEK/ERK1/2 signalling where MEK is MAPK (mitogen-activated protein kinase)/ERK kinase to eNOS and a complex mechanism including Ca2+i, phosphorylation and, possibly, intracellular trafficking.
The serine proteinase inhibitor, plasminogen activator inhibitor type-1 (PAI-1), binds to the adhesion protein vitronectin with high affinity at a site that is located directly adjacent to the ...vitronectin RGD integrin binding sequence. The binding of PAI-1 to vitronectin sterically blocks integrin access to this site and completely inhibits the binding of purified integrins to vitronectin; however, its inhibition of endothelial and smooth muscle cell adhesion to vitronectin is at most 50-75%. Because PAI-1 binds vitronectin with ∼10-100-fold higher affinity than purified integrins, we have analyzed the mechanism whereby these cells are able to overcome this obstacle. Our studies exclude proteolytic removal of PAI-1 from vitronectin as the mechanism, and show instead that cell adhesion in the presence of PAI-1 is dependent on integrin-cytoskeleton engagement. Disrupting endothelial or smooth muscle cell actin polymerization and/or focal adhesion assembly reduces cell adhesion to vitronectin in the presence of PAI-1 to levels similar to that observed for the binding of purified integrins to vitronectin. Furthermore, endothelial cell, but not smooth muscle cell adhesion to vitronectin in the presence of PAI-1 requires both polymerized microtubules and actin, further demonstrating the importance of the cytoskeleton for integrin-mediated adhesion. Finally, we show that cell adhesion in the presence of PAI-1 leads to colocalization of PAI-1 with the integrins αvβ3 and αvβ5 at the cell-matrix interface.
Pregnancy enhanced nitric oxide production by uterine artery endothelial cells (UAEC) is the result of reprogramming of both Ca(2+) and kinase signaling pathways. Using UAEC derived from pregnant ...ewes (P-UAEC), as well as COS-7 cells transiently expressing ovine endothelial nitric oxide synthase (eNOS), we investigated the role of phosphorylation of five known amino acids following treatment with physiological calcium-mobilizing agent ATP and compared with the effects of PMA (also known as TPA) alone or in combination with ATP. In P-UAEC, ATP stimulated eNOS activity and phosphorylation of eNOS S617, S635, and S1179. PMA promoted eNOS phosphorylation but without activation. PMA and ATP cotreatment attenuated ATP-stimulated activity despite no increase in phospho (p)-T497 and potentiation of p-S1179. In COS-7 cells, PMA inhibition of ATP-stimulated eNOS activity was associated with p-T497 phosphorylation. Although T497D eNOS activity was reduced to 19% of wild-type eNOS with ATP and 44% with A23187, we nonetheless observed more p-S1179 with ATP than with A23187 (3.4-fold and 1.8-fold of control, respectively). Furthermore, the S1179A eNOS mutation partly attenuated ATP- but not A23187-stimulated activity, but when combined with T497D, no further reduction of eNOS activity was observed. In conclusion, although phosphorylation of eNOS is associated with activation in P-UAEC, no single or combination of phosphorylation events predict activity changes. In COS-7 cells, phosphorylation of T497 can attenuate activity but also influences S1179 phosphorylation. We conclude that in both cell types, observed changes in phosphorylation of key residues may influence eNOS activation but are not sufficient alone to describe eNOS activation.
Commercially available human complementary DNA microarrays were used to compare differential expression in the livers of Atlantic salmon ( Salmo salar) infected with Aeromonas salmonicida and of ...healthy fish. Complementary DNA probes were prepared from total RNA isolated from livers of control salmon and infected salmon by reverse transcription in the presence of (33)P-dCTP and independently hybridized to human GENE-FILTERS GF211 microarrays. Of the 4131 known genes on the microarray, 241 spots gave clearly detectable signals using labeled RNA from the control salmon liver. Of these, 4 spots were consistently found to have a greater than 2-fold increase in infected salmon compared with controls when using the same pair of filters to generate hybridization data from triplicates. These up-regulated genes were ADP/ATP translocase (AAT2), Na(+)/K(+) ATPase, acyloxyacyl hydrolase (AOAH), and platelet-derived growth factor (PDFG-A). A BlastN search revealed an AAT2 homolog from Atlantic salmon, and a reverse transcriptase polymerase chain reaction assay using primers based on this sequence confirmed its up-regulation (approx. 1.8-fold) during early infection. This work demonstrates the feasibility of using human microarrays to facilitate the discovery of differentially expressed genes in Atlantic salmon, for which no homologous microarrays are available.
Although pregnancy is clearly associated with refractoriness to infused angiotensin II (AII) in the uteroplacental unit, there is still dispute over the mechanism by which angiotensin type 1 and type ...2 receptors (AT1R and AT2R) may mediate this response in the uterine artery. This is in large part due to incomplete knowledge of levels of AT1R and AT2R expression and function in uterine artery endothelium (UA Endo) in the nonpregnant (NP) and pregnant (P) states, combined with the disagreement on whether AII may act through release of adrenomedullary catecholamines. The authors have previously described an increase in AT1R in UA Endo but not UA vascular smooth muscle (VSM) during pregnancy as compared to the nonpregnant intact ewe. Herein they report that the pregnancy-associated increase in AT1R expression in UA Endo is regulated by ovarian steroids. Using a recently developed antibody to AT2R, the authors now show there is no change in AT2R in UA Endo or VSM associated with ovarian function, and although AT2R is not changed in UA Endo by pregnancy, there is a significant decrease observed in UA VSM at that time. The authors also examined changes in receptors in UA Endo and VSM in estrogen (E2β)-primed ewes in view of the common use of this model as a control for physiologic studies. In contrast to their findings in nonprimed nonpregnant or pregnant animals, the authors observed a significant increase in both AT1R and AT2R in UA Endo in response to the supraphysiologic priming with E2β. In order to address the possible functionality of AT1R or AT2R in UA Endo, the authors used the uterine artery endothelial cell (UAEC) model of UA endothelial cells maintained in culture to passage 4. Differences in expression of AT1R or AT2R were normalized at passage 4 in P-UAECs and NP-UAECs. Treatment with AII activated phospholipase C (PLC) in both NP- and P-UAECs but signaling through the extracellular signal-regulated kinase (ERK) pathway was dramatically enhanced in P-UAECs compared to NP-UAECs. Surprisingly, both phosphoinositol turnover and ERK2 phosphorylation responses failed to display the expected dose-responses. Inhibition of AII-stimulated ERK2 phosphorylation with antagonists DUP 753 (AT1R, 10 μM) and PD 123319 (AT2R, 10 μM) failed to selectively inhibit ERK2 phosphorylation. The authors conclude that (a) the net effect of pregnancy may be an increase in the AT1R/AT2R ratio in both UA Endo and VSM but through apparently distinct mechanisms, (b) the ovariectomized animal model is similar to the luteal state for AT1R and AT2R expression, while the E2β-primed model does not resemble the nonpregnant or pregnant state, and (c) there is a real possibility that AII may mediate its effects either through a complex AT1R-AT2R interaction or via an as-yet unidentified non-AT1, non-AT2 receptor.
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