J. Anthony Ware

Local perivascular delivery of basic fibroblast growth factor in patients undergoing coronary bypass surgery: results of a phase I randomized, double-blind, placebo-controlled trial.

BACKGROUND Angiogenesis is a promising treatment strategy for patients who are not candidates for standard revascularization, because it promotes the growth of new blood vessels in ischemic myocardium. METHODS AND RESULTS We conducted a randomized, double-blind, placebo-controlled study of basic fibroblast growth factor (bFGF; 10 or 100 microg versus placebo) delivered via sustained-release heparin-alginate microcapsules implanted in ischemic and viable but ungraftable myocardial territories in patients undergoing CABG. Twenty-four patients were randomized to 10 microg of bFGF (n=8), 100 microg of bFGF (n=8), or placebo (n=8), in addition to undergoing CABG. There were 2 operative deaths and 3 Q-wave myocardial infarctions. There were no treatment-related adverse events, and there was no rise in serum bFGF levels. Clinical follow-up was available for all patients (16.0+/-6.8 months). Three control patients had recurrent angina, 2 of whom required repeat revascularization. One patient in the 10-microg bFGF group had angina, whereas all patients in the 100-microg bFGF group remained angina-free. Stress nuclear perfusion imaging at baseline and 3 months after CABG showed a trend toward worsening of the defect size in the placebo group (20.7+/-3.7% to 23.8+/-5.7%, P=0.06), no significant change in the 10-microg bFGF group, and significant improvement in the 100-microg bFGF group (19.2+/-5.0% to 9.1+/-5.9%, P=0.01). Magnetic resonance assessment of the target ischemic zone in a subset of patients showed a trend toward a reduction in the target ischemic area in the 100-microg bFGF group (10.7+/-3.9% to 3. 7+/-6.3%, P=0.06). CONCLUSIONS This study of bFGF in patients undergoing CABG demonstrates the safety and feasibility of this mode of therapy in patients with viable myocardium that cannot be adequately revascularized.

Vascular Endothelial Growth Factor–Induced Endothelial Cell Migration and Proliferation Depend on a Nitric Oxide–Mediated Decrease in Protein Kinase Cδ Activity

Vascular endothelial growth factor (VEGF) promotes angiogenesis and endothelial cell (EC) migration and proliferation by affecting intracellular mediators, only some of which are known, distal to its receptors. Protein kinase C (PKC) participates in the function of VEGF, but the role of individual PKC isoenzymes is unknown. In this study, we tested the importance of the activity of specific PKC isoenzymes in human EC migration and proliferation in response to VEGF. PKCdelta specific activity was depressed by the addition of VEGF (by 41+/-8% [P<0.05] at 24 hours) in human umbilical vein ECs (HUVECs) and in a HUVEC-derived EC line, ECV, without changing the total amount of either protein or mRNA encoding PKCdelta. Neither basic fibroblast growth factor (FGF-2) nor serum altered PKCdelta specific activity. The VEGF-induced decrease of PKCdelta activity, which began at 8 hours after stimulation, was strongly blocked by pretreatment with the nitric oxide (NO) synthase inhibitor N(G)-monomethyl-L-arginine in HUVECs; NO release peaked within 2 hours after stimulation. An exogenous NO donor, sodium nitroprusside, also decreased PKCdelta activity. The inhibition by N(G)-monomethyl-L-arginine of VEGF-induced HUVEC migration and proliferation, but not that induced by FGF-2 or serum, suggested that the decrease in PKCdelta via NO pathway is required for VEGF-induced EC migration and proliferation. Overexpression of PKCdelta in ECV cells specifically prevented EC response to VEGF but not to FGF-2 or serum. Thus, we conclude that suppression of PKCdelta activity via a NO synthase mechanism is required for VEGF-induced EC migration and proliferation, but not for that induced by FGF-2 or serum.

Inhibition of Endothelial Cell Migration, Intercellular Communication, and Vascular Tube Formation by Thromboxane A2

The eicosanoid thromboxane A2 (TXA2) is released by activated platelets, monocytes, and the vessel wall and interacts with high affinity receptors expressed in several tissues including endothelium. Whether TXA2 might alter endothelial migration and tube formation, two determinants of angiogenesis, is unknown. Thus, we investigated the effect of the TXA2 mimetic [1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5′-heptenoic acid (IBOP) on human endothelial cell (HEC) migration and angiogenesis in vitro. IBOP stimulation inhibited HEC migration by 50% and in vitro capillary formation by 75%. These effects of IBOP were time- and concentration-dependent with an IC50 of 25 nm. IBOP did not affect integrin expression or cytoskeletal morphology of HEC. Since gap junction-mediated intercellular communication increases in migrating HEC, we determined whether IBOP might inhibit coupling or connexin expression in HEC. IBOP reduced the passage of microinjected dyes between HEC by 50%, and the effects of IBOP on migration and tube formation were mimicked by the gap junction inhibitor 18β-glycyrrhetinic acid (1 μm) with a similar time course and efficacy. IBOP (24 h) did not affect the expression or phosphorylation of connexin 43 in whole HEC lysates. Immunohistologic examination of HEC suggested that IBOP may impair functional coupling by altering the cellular distribution of gap junctions, leading to increased connexin 43 internalization. Thus, this finding that TXA2 mimetics can prevent HEC migration and tube formation, possibly by impairing intercellular communication, suggests that antagonizing TXA2 signaling might enhance vascularization of ischemic tissue.

Endothelial Cell Activation in Patients With Decompensated Heart Failure

Background—Vascular endothelial functions, other than nitric oxide (NO)–mediated control of vasomotor tone, are poorly characterized in patients with chronic heart failure (CHF). Veins and arteries are exposed to the same circulating proinflammatory mediators in patients with CHF. The present study tested whether endothelial cell activation occurs in veins of patients with decompensated CHF and whether activation, if present, subsides with return to a clinically compensated state. Methods and Results—Fifteen patients with decompensated CHF requiring transient inotropic support and 6 age-matched, healthy controls were studied. Endothelial cells and blood were collected from a forearm vein, and brachial artery flow–mediated dilation (FMD) was measured before and 24 hours after discontinuation of short-term inotropic therapy, when patients had returned to a steady compensated state. Nitrotyrosine immunoreactivity (an intracellular marker of oxidative stress), cyclooxygenase-2 (COX-2), and inducible NO synthase (iNOS) expression were significantly higher in venous endothelial cells of patients in clinical decompensation when compared with healthy subjects. Return to a compensated state resulted in a significant reduction in nitrotyrosine immunoreactivity, COX-2, and iNOS expression. Concomitantly, a significant increase in FMD and a decline in plasma total 8-isoprostane and bicycloprostaglandin E2 levels were observed. Venous endothelial NOS expression was unaffected by clinical decompensation. Conclusions—Clinical decompensation in CHF is associated with activation of the venous endothelium. Return to a compensated state after short-term inotropic therapy results in a significant reduction in endothelial nitrotyrosine formation, COX-2, and iNOS expression.

Role of Fibrinogen in Activation of Platelets by Artificial Surfaces

Activation of platelets by contact with artificial surfaces is a key event in the thromboembolic complications of prosthetic devices in contact with the blood,’*2 but the mechanism of these events is not fully understood. It is known that a film of plasma protein adsorbs on artificial materials exposed to blood and that this event precedes interaction of the surface with blood cell^.^*^ A corollary of this thesis is that the blood cells do not react directly with the underlying surface but rather with the adsorbed protein coat. The composition of the protein film and the configuration of its molecular constituents must relay to the blood elements information describing the nature of the underlying surface. Fibrinogen appears to be an important component of the adsorbed protein film and may be an essential player in the early events of platelet a c t i v a t i ~ n , ~ ~ ~ adsorbing preferentially on many surfaces in a concentration relatively higher than in the bulk plasma and participating in the platelet response to the prosthetic Fibrinogen is now recognized as an indispensible element in platelet aggregat i ~ n . ’ ~ . ’ ~ An early feature of activation of platelets by soluble agonists such as ADP or thrombin is exposure of specific membrane glycoproteins (GP IIb-IIIa), to which fibrinogen molecules bind with high According to current dogma, the divalent fibrinogen molecule is necessary for the formation of connecting bridges between contiguous platelets, perhaps in conjunction with other “adhesive prot e i n ~ , ” ’ ~ ’ ~ such as von Willebrand’s factor, thrombospondin, or fibronectin. It should be noted that fibrinogen dissolved in plasma does not induce platelet aggregation but rather acts as a cofactor in the process. Fibrinogen normally circulates in peaceful coexistence with platelets without any obvious interaction. It is claimed that platelets have membrane receptors of low affinity for fibrinogen,’”’’ which would permit reversible interactions at physiologic fibrinogen concentrations, but the prevailing view is that it is only in the “activated” platelet (i.e., the platelet stimulated by agonists such as thrombin or ADP) that fibrinogen forms a detectable complex with its glycoprotein membrane receptors, which are “masked” or otherwise unavailable in intact nonactivated circulating I f adsorbed fibrinogen were involved in some way in platelet adhesion to surfaces, perhaps by bridging the gap between platelet and surface in a manner analogous to its action in platelet aggregation, an important question would be whether prior activation of the platelet would be required or whether interaction with adsorbed fibrinogen molecules might occur without preliminary alteration of the basal platelet. It has been suggested that fibrinogen molecules adsorbed on artificial surfaces undergo conformational changes that lead platelets to regard them as “foreign” and that might even

Thromboxane A2 Receptor Signaling Inhibits Vascular Endothelial Growth Factor–Induced Endothelial Cell Differentiation and Migration

Vascular endothelial growth factor (VEGF) is an important patho-physiological mediator of angiogenesis. VEGF-induced endothelial cell (EC) migration and angiogenesis often occur in complicated environments containing multiple agents capable of modifying the response. Thromboxane (TX) A2 is released from multiple cell types and is a prime mediator of pathogenesis of many vascular diseases. Human EC express both TXA2 receptor (TP) isoforms; however, the effects of individual TP isoforms on VEGF-induced EC migration and angogenesis are unknown. We report here that the TXA2 mimetic [1S-(1&agr;, 2&bgr;(5Z), 3&agr;(1E, 3R), 4&agr;]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5′-heptenoic acid (IBOP) (100 nmol/L) is a potent antagonist (IC50 30 nmol/L) of VEGF-induced EC migration and differentiation. TP&bgr;, but not TP&agr;, expression is required for the inhibition of VEGF-induced migration and angiogenesis. IBOP costimulation suppressed nitric oxide (NO) release from VEGF-treated EC through decreased activation of Akt, eNOS, and PDK1. TP&bgr; costimulation also ablated the increase in focal adhesion formation in response to VEGF. This mechanism was characterized by decreased recruitment of focal adhesion kinase (FAK) and vinculin to the &agr;v&bgr;3 integrin and reduced FAK and Src activation in response to VEGF. Addition of NO donors together with transfection of a constitutively active Src construct could circumvent the blockade of VEGF-induced migration by TP; however, neither intervention alone was sufficient. Thus, TP stimulation appears to limit angiogenesis, at least in part, by inhibiting the pro-angiogenic cytokine VEGF. These data further support a role for antagonism of TP activation in enhancing the angiogenic response in tissues exposed to elevated TXA2 levels in which revascularization is important.

Inhibition of Protein Kinase Cα Prevents Endothelial Cell Migration and Vascular Tube Formation In Vitro and Myocardial Neovascularization In Vivo

Although protein kinase C (PKC) activation is required for endothelial cell (EC) growth, migration, adhesion, and vessel formation, the role of individual PKC isoenzymes in these events is not defined. Because PKC&agr; has been previously linked with enhanced EC migration and response to angiogenic growth factors, we characterized a specific phosphorothioate-modified 21-mer antisense PKC&agr; (AS-PKC&agr;). AS-PKC&agr; (500 nmol/L) prevented the expression of PKC&agr; protein by 90% in human ECs and did not reduce the expression of any other PKC isoenzyme. AS-PKC&agr; reduced human EC migration by 64% compared with its control oligonucleotide in a “scratch” wounding assay, and AS-PKC&agr; reduced human EC adhesion to the extracellular matrix protein vitronectin by 18%. Phosphorylation of mitogen-activated protein kinase (extracellular signal–regulated kinase 1/2) induced by vascular endothelial growth factor was inhibited by 30% in human ECs transfected with AS-PKC&agr;. Compared with control, AS-PKC&agr; also reduced the number of EC tubes formed in a 3D type I collagen gel assay by 37.5%. Finally, using an osmotic minipump, we infused AS-PKC&agr; into mice in which myocardial infarction was induced by coronary ligation and found that the oligonucleotide was primarily taken up by intramyocardial blood vessels. Compared with the results with control oligonucleotide, AS-PKC&agr; oligonucleotide inhibited the number of anti-PKC&agr;–stained blood vessels by 48% and reduced the total vessel number by 72% as well. In conclusion, the expression of PKC&agr; is required for full EC migration, adhesion to vitronectin, vascular endothelial growth factor–induced extracellular signal–regulated kinase activation, and tube formation and is likely to be of importance in myocardial angiogenesis in vivo after ischemia.

Requirement for Protein Kinase C Activation in Basic Fibroblast Growth Factor–Induced Human Endothelial Cell Proliferation

The intracellular signaling mechanisms that mediate basic fibroblast growth factor (bFGF)-induced angiogenesis have not been fully identified. In particular, whether activation of the intracellular enzyme protein kinase C (PKC) is necessary or sufficient for bFGF-induced mitogenesis of human endothelial cells is not clear. Accordingly, the effect of bFGF stimulation on the Ca2+ increase and PKC activity of normal human endothelial cells (HEC) was studied, as was the effect of inhibition of PKC and the distribution of PKC isoenzymes in these cells. The addition of bFGF to cultured HEC increased overall PKC activity in the absence of an increase in intracellular Ca2+ and markedly stimulated their proliferation, as did the addition of PKC-activating phorbol esters. bFGF-induced proliferation was prevented by the PKC inhibitors chelerythrine and H-7 and by downregulation of PKC after prolonged incubation with phorbol esters. In contrast, these inhibitors did not prevent HEC proliferation induced by epidermal growth factor. Because of the failure of bFGF to increase Ca2+, we determined whether bFGF-induced proliferation could be mediated by novel or atypical PKC isoenzymes (which are not regulated by Ca2+). Investigation of the isoenzyme distribution of confluent and subconfluent HEC by immunoblotting, Northern transfer analysis, and polymerase chain reaction of reverse-transcribed RNA revealed the presence of several novel and atypical isoenzymes (PKC-delta, -eta, -theta, and -zeta) as well as small amounts of the conventional (Ca(2+)-regulated) isoenzymes PKC-alpha and -beta. Activation of PKC by bFGF, in the absence of an increase in intracellular Ca2+, suggests that one or more of these Ca(2+)-independent PKC isoenzymes are both necessary and sufficient for HEC proliferation after bFGF.

Reversal of Angiogenesis In Vitro, Induction of Apoptosis, and Inhibition of Akt Phosphorylation in Endothelial Cells by Thromboxane A2

Thromboxane A(2) (TxA(2)) causes platelet aggregation, vasoconstriction, and inhibition of endothelial cell (EC) migration and prevents vascular tube formation via its specific receptors (TP), of which there are two isoforms (TPalpha and TPbeta), both expressed in human ECs. In this study, we demonstrate that the TxA(2) mimetic IBOP increases apoptosis of human ECs and inhibits the phosphorylation of Akt kinase, an intracellular mediator required for cell survival. Treatment with IBOP destroyed EC networks formed on a basement membrane matrix in vitro. To distinguish the role of the TP isoforms, each isoform was expressed in TP-null ECs to create TPalpha and TPbeta ECs. IBOP induced apoptosis and inhibited phosphorylation of Akt kinase in both TPalpha and TPbeta. IBOP increased cAMP levels in TPalpha but not in TPbeta. Apoptosis induced by IBOP in TPalpha was not affected by either the adenylyl cyclase activator forskolin or the protein kinase A inhibitor 14-22 amide or H-89, whereas that in TPbeta was suppressed by forskolin and enhanced by the protein kinase A inhibitor 14-22 amide or H-89, suggesting that the TP isoforms differ in their signal pathways in mediating apoptosis. In conclusion, apoptosis may be the mechanism by which TxA(2)-mediated destruction of vascular structures in ECs occurs; although both TP isoforms induce apoptosis, possibly via inhibiting Akt phosphorylation, the signaling differs in each isoform, in that activation of the adenylyl cyclase pathway prevents apoptosis caused by TPbeta, but not by TPalpha, stimulation.

Intravenous Cocaine Induces Platelet Activation in the Conscious Dog

BACKGROUND Cocaine consumption has been associated with thrombosis of coronary and peripheral arteries. Since cocaine has been found to induce platelet activation in vitro, we sought to establish whether cocaine induced platelet activation in vivo. METHODS AND RESULTS Chronically instrumented, conscious dogs were infused with cocaine (1 mg/kg), norepinephrine (0.2 to 0.4 mg/kg), or saline intravenously over 1 minute. Activated canine platelets were identified in whole blood collected from an indwelling aortic catheter by flow cytometric detection of the binding of a monoclonal antibody directed against the activation-dependent antigen P-selectin. Infusion of cocaine resulted in an elevation of mean arterial pressure (91 +/- 3 to 128 +/- 7 mm Hg [P < .01]) and heart rate (87 +/- 9 to 125 +/- 11 beats per minute [P < .01]). A similar change (P = NS) in mean arterial pressure followed norepinephrine infusion (100 +/- 5 to 137 +/- 13 mm Hg [P < .04]), whereas saline infusion had no effect. Cocaine resulted in a substantial but delayed increase in platelet P-selectin expression (14 +/- 7% [P < .08], 31 +/- 12% [P < .04], and 55 +/- 22% [P < .04] at 17, 22, and 27 minutes after drug infusion, respectively). The magnitude of this increase was similar to that found in blood treated ex vivo with the agonists ADP or PAF (23 +/- 7% and 53 +/- 15%, respectively). No significant increase in P-selectin expression was detected in the blood of animals that received norepinephrine or saline. Serum cocaine concentrations were highest immediately after infusion (538 +/- 55 ng/mL at 2 minutes) but declined rapidly (185 +/- 22 and 110 +/- 25 ng/mL at 17 and 32 minutes after infusion); in contrast, the increase in benzoylecgonine concentrations was delayed (from < 25 ng/mL in all but one animal [34 ng/mL] at 2 minutes to 46 +/- 4 and 71 +/- 11 ng/mL at 17 and 32 minutes, respectively, after infusion). CONCLUSIONS Intravenous cocaine induces activation of individual circulating platelets; this effect is not reproduced by infusion of norepinephrine at doses sufficient to exert similar hemodynamic effects. The delay in detection of activated platelets after treatment with cocaine may result from the adhesion and subsequent detachment of activated platelets; alternatively, cocaine metabolites, rather than the drug itself, may induce platelet activation.