Enhanced Reendothelialization and Thrombosis Prevention with a New Drug-Eluting Stent.

PURPOSE: The objective of the study is to test the efficacy of cyclopentenyl cytosine (CPEC)-coated stents on blocking artery stenosis, promoting reendothelialization, and reducing thrombosis. METHODS: Scanning electron microscopy was employed to observe the morphological characteristics of stents coated with a mixture of CPEC and poly(lactic-co-glycolic acid) (PLGA) copolymer. PLGA has been used in various Food and Drug Administration (FDA)-approved therapeutic devices. In vitro release of CPEC was tested to measure the dynamic drug elution. Comparison between CPEC- and everolimus-coated stents on neointimal formation and thrombosis formation was conducted after being implanted into the human internal mammary artery and grafted to the mouse aorta. RESULTS: Optimization in stent coating resulted in uniform and consistent coating with minimal variation. In vitro drug release tests demonstrated a gradual and progressive discharge of CPEC. CPEC- or everolimus-coated stents caused much less stenosis than bare-metal stents. However, CPEC stent-implanted arteries exhibited enhanced reendothelialization compared to everolimus stents. Mechanistically, CPEC-coated stents reduced the proliferation of vascular smooth muscle cells while simultaneously promoting reendothelialization. More significantly, unlike everolimus-coated stents, CPEC-coated stents showed a significant reduction in thrombosis formation even in the absence of ongoing anticoagulant treatment. CONCLUSIONS: The study establishes CPEC-coated stent as a promising new device for cardiovascular interventions. By enhancing reendothelialization and preventing thrombosis, CPEC offers advantages over conventional approaches, including the elimination of the need for anti-clogging drugs, which pave the way for improved therapeutic outcomes and management of atherosclerosis-related medical procedures.

PAI-1 Regulates the Cytoskeleton and Intrinsic Stiffness of Vascular Smooth Muscle Cells.

BACKGROUND: Plasma concentration of PAI-1 (plasminogen activator inhibitor-1) correlates with arterial stiffness. Vascular smooth muscle cells (SMCs) express PAI-1, and the intrinsic stiffness of SMCs is a major determinant of total arterial stiffness. We hypothesized that PAI-1 promotes SMC stiffness by regulating the cytoskeleton and that pharmacological inhibition of PAI-1 decreases SMC and aortic stiffness. METHODS: PAI-039, a specific inhibitor of PAI-1, and small interfering RNA were used to inhibit PAI-1 expression in cultured human SMCs. Effects of PAI-1 inhibition on SMC stiffness, F-actin (filamentous actin) content, and cytoskeleton-modulating enzymes were assessed. WT (wild-type) and PAI-1-deficient murine SMCs were used to determine PAI-039 specificity. RNA sequencing was performed to determine the effects of PAI-039 on SMC gene expression. In vivo effects of PAI-039 were assessed by aortic pulse wave velocity. RESULTS: PAI-039 significantly reduced intrinsic stiffness of human SMCs, which was accompanied by a significant decrease in cytoplasmic F-actin content. PAI-1 gene knockdown also decreased cytoplasmic F-actin. PAI-1 inhibition significantly increased the activity of cofilin, an F-actin depolymerase, in WT murine SMCs, but not in PAI-1-deficient SMCs. RNA-sequencing analysis suggested that PAI-039 upregulates AMPK (AMP-activated protein kinase) signaling in SMCs, which was confirmed by Western blotting. Inhibition of AMPK prevented activation of cofilin by PAI-039. In mice, PAI-039 significantly decreased aortic stiffness and tunica media F-actin content without altering the elastin or collagen content. CONCLUSIONS: PAI-039 decreases intrinsic SMC stiffness and cytoplasmic stress fiber content. These effects are mediated by AMPK-dependent activation of cofilin. PAI-039 also decreases aortic stiffness in vivo. These findings suggest that PAI-1 is an important regulator of the SMC cytoskeleton and that pharmacological inhibition of PAI-1 has the potential to prevent and treat cardiovascular diseases involving arterial stiffening.

In vitro biological responses of plasma nanocoatings for coronary stent applications.

In-stent restenosis and thrombosis remain to be long-term challenges in coronary stenting procedures. The objective of this study was to evaluate the in vitro biological responses of trimethylsilane (TMS) plasma nanocoatings modified with NH3 /O2 (2:1 molar ratio) plasma post-treatment (TMS + NH3 /O2 nanocoatings) on cobalt chromium (CoCr) alloy L605 coupons, L605 stents, and 316L stainless steel (SS) stents. Surface properties of the plasma nanocoatings with up to 2-year aging time were characterized by wettability assessment and x-ray photoelectron spectroscopy (XPS). It was found that TMS + NH3 /O2 nanocoatings had a surface composition of 41.21 ± 1.06 at% oxygen, 31.90 ± 1.08 at% silicon, and 24.12 ± 1.7 at% carbon, and very small but essential amount of 2.77 ± 0.18 at% nitrogen. Surface chemical stability of the plasma coatings was noted with persistent O/Si atomic ratio of 1.292-1.413 and N/Si atomic ratio of ~0.087 through 2 years. The in vitro biological responses of plasma nanocoatings were studied by evaluating the cell proliferation and migration of porcine coronary artery endothelial cells (PCAECs) and smooth muscle cells (PCASMCs). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay results revealed that, after 7-day incubation, TMS + NH3 /O2 nanocoatings maintained a similar level of PCAEC proliferation while showing a decrease in the viability of PCASMCs by 73 ± 19% as compared with uncoated L605 surfaces. Cell co-culture of PCAECs and PCASMCs results showed that, the cell ratio of PCAEC/PCASMC on TMS + NH3 /O2 nanocoating surfaces was 1.5-fold higher than that on uncoated L605 surfaces, indicating enhanced selectivity for promoting PCAEC growth. Migration test showed comparable PCAEC migration distance for uncoated L605 and TMS + NH3 /O2 nanocoatings. In contrast, PCASMC migration distance was reduced nearly 8.5-fold on TMS + NH3 /O2 nanocoating surfaces as compared to the uncoated L605 surfaces. Platelet adhesion test using porcine whole blood showed lower adhered platelets distribution (by 70 ± 16%), reduced clotting attachment (by 54 ± 12%), and less platelet activation on TMS + NH3 /O2 nanocoating surfaces as compared with the uncoated L605 controls. It was further found that, under shear stress conditions of simulated blood flow, TMS + NH3 /O2 nanocoating significantly inhibited platelet adhesion compared to the uncoated 316L SS stents and TMS nanocoated 316L SS stents. These results indicate that TMS + NH3 /O2 nanocoatings are very promising in preventing both restenosis and thrombosis for coronary stent applications.

A Biocompatibility Study of Plasma Nanocoatings onto Cobalt Chromium L605 Alloy for Cardiovascular Stent Applications.

The objective of this study was to evaluate the biocompatibility of trimethylsilane (TMS) plasma nanocoatings modified with NH3/O2 (2:1 molar ratio) plasma post-treatment onto cobalt chromium (CoCr) L605 alloy coupons and stents for cardiovascular stent applications. Biocompatibility of plasma nanocoatings was evaluated by coating adhesion, corrosion behavior, ion releasing, cytotoxicity, and cell proliferation. Surface chemistry and wettability were studied to understand effects of surface properties on biocompatibility. Results show that NH3/O2 post-treated TMS plasma nanocoatings are hydrophilic with water contact angle of 48.5° and have a typical surface composition of O (39.39 at.%), Si (31.92 at.%), C (24.12 at.%), and N (2.77 at.%). The plasma nanocoatings were conformal to substrate surface topography and had excellent adhesion to the alloy substrates, as assessed by tape test (ASTM D3359), and showed no cracking or peeling off L605 stent surfaces after dilation. The plasma nanocoatings also improve the corrosion resistance of CoCr L605 alloy by increasing corrosion potential and decreasing corrosion rates with no pitting corrosion and no mineral adsorption layer. Ion releasing test revealed that Co, Cr, and Ni ion concentrations were reduced by 64-79%, 67-69%, and 57-72%, respectively, in the plasma-nanocoated L605 samples as compared to uncoated L605 control samples. The plasma nanocoatings showed no sign of cytotoxicity from the test results according to ISO 10993-05 and 10993-12. Seven-day cell culture demonstrated that, in comparison with the uncoated L605 control surfaces, the plasma nanocoating surfaces showed 62 ± 7.3% decrease in porcine coronary artery smooth muscle cells (PCASMCs) density and had comparable density of porcine coronary artery endothelial cells (PCAECs). These results suggest that TMS plasma nanocoatings with NH3/O2 plasma post-treatment possess the desired biocompatibility for stent applications and support the hypothesis that nanocoated stents could be very effective for in-stent restenosis prevention.

CagA+ Helicobacter pylori, Not CagA- Helicobacter pylori, Infection Impairs Endothelial Function Through Exosomes-Mediated ROS Formation.

Background: Helicobacter pylori (H. pylori) infection increases the risk for atherosclerosis, and ROS are critical to endothelial dysfunction and atherosclerosis. CagA is a major H. pylori virulence factor associated with atherosclerosis. The present study aimed to test the hypothesis that CagA+ H. pylori effectively colonizes gastric mucosa, and CagA+ H. pylori, but not CagA- H. pylori, infection impairs endothelial function through exosomes-mediated ROS formation. Methods: C57BL/6 were used to determine the colonization ability of CagA+ H. pylori and CagA- H. pylori. ROS production, endothelial function of thoracic aorta and atherosclerosis were measured in CagA+ H. pylori and CagA- H. pylori infected mice. Exosomes from CagA+ H. pylori and CagA- H. pylori or without H. pylori infected mouse serum or GES-1 were isolated and co-cultured with bEND.3 and HUVECs to determine how CagA+ H. pylori infection impairs endothelial function. Further, GW4869 was used to determine if CagA+ H. pylori infection could lead to endothelial dysfunction and atherosclerosis through an exosomes-mediated mechanism. Results: CagA+ H. pylori colonized gastric mucosa more effectively than CagA- H. pylori in mice. CagA+ H. pylori, not CagA- H. pylori, infection significantly increased aortic ROS production, decreased ACh-induced aortic relaxation, and enhanced early atherosclerosis formation, which were prevented with N-acetylcysteine treatment. Treatment with CagA-containing exosomes significantly increased intracellular ROS production in endothelial cells and impaired their function. Inhibition of exosomes secretion with GW4869 effectively prevented excessive aortic ROS production, endothelial dysfunction, and atherosclerosis in mice with CagA+ H. pylori infection. Conclusion: These data suggest that CagA+ H. pylori effectively colonizes gastric mucosa, impairs endothelial function, and enhances atherosclerosis via exosomes-mediated ROS formation in mice.

Fish Oil Supplements for Prevention of Cardiovascular Disease: The Jury Is Still Out: CON: Fish Oil is Useful to Prevent or Treat Cardiovascular Disease.

Consumption of oily fish high in omega-3 fatty acids (n-3FAs) is strongly associated with reduced risk of adverse cardiovascular events. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the n-3FAs in fish oil believed to confer its beneficial effects. Over the past two decades, multiple clinical trials have been conducted to test the hypothesis that encapsulated EPA and DHA supplements improve cardiovascular outcomes in patients with established cardiovascular disease or at risk of developing it. Over the same time period, over-the-counter fish oil supplements have become a multi-billion-dollar industry. In this article, we briefly review available clinical trial data involving EPA and DHA supplementation. Based on currently available information, we conclude that combination capsules containing EPA and DHA should not be used to reduce cardiovascular risk. Some studies suggest that EPA as stand-alone therapy decreases cardiovascular risk. Nevertheless, we advocate a restrictive approach to using EPA to improve cardiovascular outcomes.

Janus Kinase 3 Deficiency Promotes Vascular Reendothelialization-Brief Report.

[Figure: see text].

Elevated postischemic tissue injury and leukocyte-endothelial adhesive interactions in mice with global deficiency in caveolin-2: role of PAI-1.

Ischemia/reperfusion (I/R)-induced rapid inflammation involving activation of leukocyte-endothelial adhesive interactions and leukocyte infiltration into tissues is a major contributor to postischemic tissue injury. However, the molecular mediators involved in this pathological process are not fully known. We have previously reported that caveolin-2 (Cav-2), a protein component of plasma membrane caveolae, regulated leukocyte infiltration in mouse lung carcinoma tumors. The goal of the current study was to examine if Cav-2 plays a role in I/R injury and associated acute leukocyte-mediated inflammation. Using a mouse small intestinal I/R model, we demonstrated that I/R downregulates Cav-2 protein levels in the small bowel. Further study using Cav-2-deficient mice revealed aggravated postischemic tissue injury determined by scoring of villi length in H&E-stained tissue sections, which correlated with increased numbers of MPO-positive tissue-infiltrating leukocytes determined by IHC staining. Intravital microscopic analysis of upstream events relative to leukocyte transmigration and tissue infiltration revealed that leukocyte-endothelial cell adhesive interactions in postcapillary venules, namely leukocyte rolling and adhesion were also enhanced in Cav-2-deficient mice. Mechanistically, Cav-2 deficiency increased plasminogen activator inhibitor-1 (PAI-1) protein levels in the intestinal tissue and a pharmacological inhibition of PAI-1 had overall greater inhibitory effect on both aggravated I/R tissue injury and enhanced leukocyte-endothelial interactions in postcapillary venules in Cav-2-deficient mice. In conclusion, our data suggest that Cav-2 protein alleviates tissue injury in response to I/R by dampening PAI-1 protein levels and thereby reducing leukocyte-endothelial adhesive interactions.NEW & NOTEWORTHY The role of caveolin-2 in regulating ischemia/reperfusion (I/R) tissue injury and the mechanisms underlying its effects are unknown. This study uses caveolin-2-deficient mouse and small intestinal I/R injury models to examine the role of caveolin-2 in the leukocyte-dependent reperfusion injury. We demonstrate for the first time that caveolin-2 plays a protective role from the I/R-induced leukocyte-dependent reperfusion injury by reducing PAI-1 protein levels in intestinal tissue and leukocyte-endothelial adhesive interactions in postcapillary venules.

Drug Targeting of Plasminogen Activator Inhibitor-1 Inhibits Metabolic Dysfunction and Atherosclerosis in a Murine Model of Metabolic Syndrome.

OBJECTIVE: Enhanced expression of PAI-1 (plasminogen activator inhibitor-1) has been implicated in atherosclerosis formation in humans with obesity and metabolic syndrome. However, little is known about the effects of pharmacological targeting of PAI-1 on atherogenesis. This study examined the effects of pharmacological PAI-1 inhibition on atherosclerosis formation in a murine model of obesity and metabolic syndrome. Approach and Results: LDL receptor-deficient (ldlr-/-) mice were fed a Western diet high in cholesterol, fat, and sucrose to induce obesity, metabolic dysfunction, and atherosclerosis. Western diet triggered significant upregulation of PAI-1 expression compared with normal diet controls. Addition of a pharmacological PAI-1 inhibitor (either PAI-039 or MDI-2268) to Western diet significantly inhibited obesity and atherosclerosis formation for up to 24 weeks without attenuating food consumption. Pharmacological PAI-1 inhibition significantly decreased macrophage accumulation and cell senescence in atherosclerotic plaques. Recombinant PAI-1 stimulated smooth muscle cell senescence, whereas a PAI-1 mutant defective in LRP1 (LDL receptor-related protein 1) binding did not. The prosenescent effect of PAI-1 was blocked by PAI-039 and R2629, a specific anti-LRP1 antibody. PAI-039 significantly decreased visceral adipose tissue inflammation, hyperglycemia, and hepatic triglyceride content without altering plasma lipid profiles. CONCLUSIONS: Pharmacological targeting of PAI-1 inhibits atherosclerosis in mice with obesity and metabolic syndrome, while inhibiting macrophage accumulation and cell senescence in atherosclerotic plaques, as well as obesity-associated metabolic dysfunction. PAI-1 induces senescence of smooth muscle cells in an LRP1-dependent manner. These results help to define the role of PAI-1 in atherosclerosis formation and suggest a new plasma-lipid-independent strategy for inhibiting atherogenesis.

Overproduction of endothelin-1 impairs glucose tolerance but does not promote visceral adipose tissue inflammation or limit metabolic adaptations to exercise.

Endothelin-1 (ET-1) is a potent vasoconstrictor and proinflammatory peptide that is upregulated in obesity. Herein, we tested the hypothesis that ET-1 signaling promotes visceral adipose tissue (AT) inflammation and disrupts glucose homeostasis. We also tested if reduced ET-1 is a required mechanism by which exercise ameliorates AT inflammation and improves glycemic control in obesity. We found that 1) diet-induced obesity, AT inflammation, and glycemic dysregulation were not accompanied by significantly increased levels of ET-1 in AT or circulation in wild-type mice and that endothelial overexpression of ET-1 and consequently increased ET-1 levels did not cause AT inflammation yet impaired glucose tolerance; 2) reduced AT inflammation and improved glucose tolerance with voluntary wheel running was not associated with decreased levels of ET-1 in AT or circulation in obese mice nor did endothelial overexpression of ET-1 impede such exercise-induced metabolic adaptations; 3) chronic pharmacological blockade of ET-1 receptors did not suppress AT inflammation in obese mice but improved glucose tolerance; and 4) in a cohort of human subjects with a wide range of body mass indexes, ET-1 levels in AT, or circulation were not correlated with markers of inflammation in AT. In aggregate, we conclude that ET-1 signaling is not implicated in the development of visceral AT inflammation but promotes glucose intolerance, thus representing an important therapeutic target for glycemic dysregulation in conditions characterized by hyperendothelinemia. Furthermore, we show that the salutary effects of exercise on AT and systemic metabolic function are not contingent on the suppression of ET-1 signaling.

Targeting TRAF3IP2 by Genetic and Interventional Approaches Inhibits Ischemia/Reperfusion-induced Myocardial Injury and Adverse Remodeling.

Re-establishing blood supply is the primary goal for reducing myocardial injury in subjects with ischemic heart disease. Paradoxically, reperfusion results in nitroxidative stress and a marked inflammatory response in the heart. TRAF3IP2 (TRAF3 Interacting Protein 2; previously known as CIKS or Act1) is an oxidative stress-responsive cytoplasmic adapter molecule that is an upstream regulator of both IκB kinase (IKK) and c-Jun N-terminal kinase (JNK), and an important mediator of autoimmune and inflammatory responses. Here we investigated the role of TRAF3IP2 in ischemia/reperfusion (I/R)-induced nitroxidative stress, inflammation, myocardial dysfunction, injury, and adverse remodeling. Our data show that I/R up-regulates TRAF3IP2 expression in the heart, and its gene deletion, in a conditional cardiomyocyte-specific manner, significantly attenuates I/R-induced nitroxidative stress, IKK/NF-κB and JNK/AP-1 activation, inflammatory cytokine, chemokine, and adhesion molecule expression, immune cell infiltration, myocardial injury, and contractile dysfunction. Furthermore, Traf3ip2 gene deletion blunts adverse remodeling 12 weeks post-I/R, as evidenced by reduced hypertrophy, fibrosis, and contractile dysfunction. Supporting the genetic approach, an interventional approach using ultrasound-targeted microbubble destruction-mediated delivery of phosphorothioated TRAF3IP2 antisense oligonucleotides into the LV in a clinically relevant time frame significantly inhibits TRAF3IP2 expression and myocardial injury in wild type mice post-I/R. Furthermore, ameliorating myocardial damage by targeting TRAF3IP2 appears to be more effective to inhibiting its downstream signaling intermediates NF-κB and JNK. Therefore, TRAF3IP2 could be a potential therapeutic target in ischemic heart disease.

Pharmacological Targeting of Plasminogen Activator Inhibitor-1 Decreases Vascular Smooth Muscle Cell Migration and Neointima Formation.

OBJECTIVE: Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that promotes and inhibits cell migration, plays a complex and important role in adverse vascular remodeling. Little is known about the effects of pharmacological PAI-1 inhibitors, an emerging drug class, on migration of vascular smooth muscle cells (SMCs) and endothelial cells (ECs), crucial mediators of vascular remodeling. We investigated the effects of PAI-039 (tiplaxtinin), a specific PAI-1 inhibitor, on SMC and EC migration in vitro and vascular remodeling in vivo. APPROACH AND RESULTS: PAI-039 inhibited SMC migration through collagen gels, including those supplemented with vitronectin and other extracellular matrix proteins, but did not inhibit migration of PAI-1-deficient SMCs, suggesting that its antimigratory effects were PAI-1-specific and physiologically relevant. However, PAI-039 did not inhibit EC migration. PAI-039 inhibited phosphorylation and nuclear translocation of signal transducers and activators of transcription-1 in SMCs, but had no discernable effect on signal transducer and activator of transcription-1 signaling in ECs. Expression of low-density lipoprotein receptor-related protein 1, a motogenic PAI-1 receptor that activates Janus kinase/signal transducers and activators of transcription-1 signaling, was markedly lower in ECs than in SMCs. Notably, PAI-039 significantly inhibited intimal hyperplasia and inflammation in murine models of adverse vascular remodeling, but did not adversely affect re-endothelialization after endothelium-denuding mechanical vascular injury. CONCLUSIONS: PAI-039 inhibits SMC migration and intimal hyperplasia, while having no inhibitory effect on ECs, which seems to be because of differences in PAI-1-dependent low-density lipoprotein receptor-related protein 1/Janus kinase/signal transducer and activator of transcription-1 signaling between SMCs and ECs. These findings suggest that PAI-1 may be an important therapeutic target in obstructive vascular diseases characterized by neointimal hyperplasia.

Laminin Receptor-Avid Nanotherapeutic EGCg-AuNPs as a Potential Alternative Therapeutic Approach to Prevent Restenosis.

In our efforts to develop new approaches to treat and prevent human vascular diseases, we report herein our results on the proliferation and migration of human smooth muscles cells (SMCs) and endothelial cells (ECs) using epigallocatechin-3-gallate conjugated gold nanoparticles (EGCg-AuNPs) as possible alternatives to drug coated stents. Detailed in vitro stability studies of EGCg-AuNPs in various biological fluids, affinity and selectivity towards SMCs and ECs have been investigated. The EGCg-AuNPs showed selective inhibitory efficacy toward the migration of SMCs. However, the endothelial cells remained unaffected under similar experimental conditions. The cellular internalization studies have indicated that EGCg-AuNPs internalize into the SMCs and ECs within short periods of time through laminin receptor mediated endocytosis mode. Favorable toxicity profiles and selective affinity toward SMCs and ECs suggest that EGCg-AuNPs may provide attractive alternatives to drug coated stents and therefore offer new therapeutic approaches in treating cardiovascular diseases.

The Nox1/4 Dual Inhibitor GKT137831 or Nox4 Knockdown Inhibits Angiotensin-II-Induced Adult Mouse Cardiac Fibroblast Proliferation and Migration. AT1 Physically Associates With Nox4.

Both oxidative stress and inflammation contribute to chronic hypertension-induced myocardial fibrosis and adverse cardiac remodeling. Here we investigated whether angiotensin (Ang)-II-induced fibroblast proliferation and migration are NADPH oxidase (Nox) 4/ROS and IL-18 dependent. Our results show that the potent induction of mouse cardiac fibroblast (CF) proliferation and migration by Ang-II is markedly attenuated by Nox4 knockdown and the Nox inhibitor DPI. Further, Nox4 knockdown and DPI pre-treatment attenuated Ang-II-induced IL-18, IL-18Rα and collagen expression, and MMP9 and LOX activation. While neutralization of IL-18 blunted Ang-II-induced CF proliferation and migration, knockdown of MMP9 attenuated CF migration. The antioxidant NAC and the cell-permeable SOD mimetics Tempol, MnTBAP, and MnTMPyP attenuated oxidative stress and inhibited CF proliferation and migration. The Nox1/Nox4 dual inhibitor GKT137831 also blunted Ang-II-induced H2 O2 production and CF proliferation and migration. Further, AT1 bound Nox4, and Ang-II enhanced their physical association. Notably, GKT137831 attenuated the AT1/Nox4 interaction. These results indicate that Ang-II induces CF proliferation and migration in part via Nox4/ROS-dependent IL-18 induction and MMP9 activation, and may involve AT1/Nox4 physical association. Thus, either (i) neutralizing IL-18, (ii) blocking AT1/Nox4 interaction or (iii) use of the Nox1/Nox4 inhibitor GKT137831 may have therapeutic potential in chronic hypertension-induced adverse cardiac remodeling.

Bevacizumab promotes venous thromboembolism through the induction of PAI-1 in a mouse xenograft model of human lung carcinoma.

BACKGROUND: An increased incidence of venous thromboembolism (VTE) is associated with anti-vascular endothelial growth factor (VEGF) treatment in cancer. However, the mechanism underlying this effect remains elusive. In this study, we examined the effect of bevacizumab, a humanized monoclonal antibody against VEGF-A, on VTE in a murine xenograft A549 cell tumor model. METHODS: Inferior vena cava stenosis model and FeCl3-induced saphenous vein thrombosis model were performed in a mouse xenograft models of human lung adenocarcinoma. RESULTS: We found that treatment with bevacizumab significantly increased the thrombotic response to inferior vena cava obstruction and femoral vein injury. Plasminogen activator inhibitor (PAI-1) expression in tumors, plasma, and thrombi was significantly increased by bevacizumab. However, bevacizumab did not enhance VTE in PAI-1-deficient mice, suggesting that PAI-1 is a major mediator of bevacizumab's prothrombotic effect. VEGF inhibited expression of PAI-1 by A549 cells, and this effect was neutralized by bevacizumab, suggesting that bevacizumab increases PAI-1 expression in vivo by blocking the inhibitory effect of VEGF on PAI-1 expression by tumor cells. Pharmacological inhibition of PAI-1 with PAI-039 blocked bevacizumab-induced venous thrombosis. CONCLUSION: Collectively, these findings indicate that PAI-1 plays a role in VTE associated with antiangiogenic therapy and the inhibition of PAI-1 shows efficacy as a therapeutic strategy for the prevention of bevacizumab-associated VTE.

Plasminogen activator inhibitor-1 inhibits angiogenic signaling by uncoupling vascular endothelial growth factor receptor-2-αVß3 integrin cross talk.

OBJECTIVE: Plasminogen activator inhibitor-1 (PAI-1) regulates angiogenesis via effects on extracellular matrix proteolysis and cell adhesion. However, no previous study has implicated PAI-1 in controlling vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that PAI-1 downregulates VEGF receptor-2 (VEGFR-2) activation by inhibiting a vitronectin-dependent cooperative binding interaction between VEGFR-2 and αVß3. APPROACH AND RESULTS: We studied effects of PAI-1 on VEGF signaling in human umbilical vein endothelial cells. PAI-1 inhibited VEGF-induced phosphorylation of VEGFR-2 in human umbilical vein endothelial cells grown on vitronectin, but not on fibronectin or collagen. PAI-1 inhibited the binding of VEGFR-2 to ß3 integrin, VEGFR-2 endocytosis, and intracellular signaling pathways downstream of VEGFR-2. The anti-VEGF effect of PAI-1 was mediated by 2 distinct pathways, one requiring binding to vitronectin and another requiring binding to very low-density lipoprotein receptor. PAI-1 inhibited VEGF-induced angiogenesis in vitro and in vivo, and pharmacological inhibition of PAI-1 promoted collateral arteriole development and recovery of hindlimb perfusion after femoral artery interruption. CONCLUSIONS: PAI-1 inhibits activation of VEGFR-2 by VEGF by disrupting a vitronectin-dependent proangiogenic binding interaction involving αVß3 and VEGFR-2. These results broaden our understanding of the roles of PAI-1, vitronectin, and endocytic receptors in regulating VEGFR-2 activation and suggest novel therapeutic strategies for regulating VEGF signaling.