Angiogenesis: where do we stand now?
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Cardiovascular Diseases↗
Biomedical subjects
Michael Simons
Publications and source records attributed to Michael Simons.
Syndecans: new kids on the signaling block.
Cell-associated proteoglycans provide highly complex and sophisticated systems to control interactions of extracellular cell matrix components and soluble ligands with the cell surface. Syndecans, a conserved family of heparan- and chondroitin-sulfate carrying transmembrane proteins, are emerging as central players in these interactions. Recent studies have demonstrated the essential role of syndecans in modulating cellular signaling in embryonic development, tumorigenesis, and angiogenesis. In this review, we focus on new advances in our understanding of syndecan-mediated cell signaling.
Animals↗
Growth factor-induced therapeutic angiogenesis in the heart: protein therapy.
Therapeutic angiogenesis, stimulated growth of new vasculature to compensate for tissue ischemia, remains an unfulfilled promise. After nearly a decade of trials, the development of drugs capable of stimulating revascularization of underperfused tissues remains an exciting but unrealized goal in cardiovascular therapeutics. This review will summarize experiences in therapeutic angiogenesis studies employing protein therapies and will draw conclusions based on our current understanding of biological processes of new vessel growth.
Adult↗
Live 3D echo guidance of catheter-based endomyocardial injection.
Local delivery of therapeutic agents into the myocardium is limited by suboptimal imaging. We evaluated the feasibility and accuracy of live 3D echo to guide left ventricular endomyocardial injection. An intramyocardial injection catheter was positioned in the left ventricle in five healthy Yorkshire pigs using fluoroscopy. All other catheter manipulations were performed with live biplane and 3D echo guidance. In each animal, a total of 12 endomyocardial injections (volume, 50-100 microl) of echo contrast mixed with blue tissue dye were performed. Four injections, 10 mm apart, were directed to three myocardial target zones: the anterior septum at the mitral valve level (zone 1); the posterolateral wall between the heads of the papillary muscles (zone 2); and the apex (zone 3). The injections were aimed to form a transverse line in zones 1 and 2 and an inverted triangular pyramid in zone 3. The animals were sacrificed, the hearts were inspected and the left ventricular endocardium was examined to create a map of injection marks. Success, defined as a visible injection of tissue dye, was 95%, and accuracy, defined as an injection into the target zone, was 83%. There was no significant difference in accuracy between the zones. Live 3D echo can successfully guide endomyocardial injections by accurately targeting specific myocardial zones, verifying catheter apposition and, when combined with echo contrast, providing real-time visualization of injectate deposition.
Animals↗
Characterization of synectin expression and promoter activity.
Synectin, a PDZ2 protein, binds to a number of plasma membrane receptors. We characterized synectin promoter and carried out a detailed expression survey. The protein is widely distributed in adult cells and tissues. Expression in the mouse embryo is apparent at day 4.5, peaks at day 12.5 and declines to its adult levels by day 18.5. The level of synectin transcription was minimally affected by exposure to cytokines, growth factors or hypoxia. In summary, synectin's early appearance, constant nature, and ubiquitous distribution suggest that it may be a key structural protein involved in mediation of outside-in signaling.
3T3 Cells↗
Angiogenesis-dependent and independent phases of intimal hyperplasia.
BACKGROUND: Neointimal vascular smooth muscle cell (VSMC) proliferation is a primary cause of occlusive vascular disease, including atherosclerosis, restenosis after percutaneous interventions, and bypass graft stenosis. Angiogenesis is implicated in the progression of early atheromatous lesions in animal models, but its role in neointimal VSMC proliferation is undefined. Because percutaneous coronary interventions result in induction of periadventitial angiogenesis, we analyzed the role of this process in neointima formation. METHODS AND RESULTS: Local injury to the arterial wall in 2 different animal models induced periadventitial angiogenesis and neointima formation. Application of angiogenesis stimulators vascular endothelial growth factor (VEGF-A165) or a proline/arginine-rich peptide (PR39) to the adventitia of the injured artery induced a marked increase in neointimal thickening beyond that seen with injury alone in both in vivo models. Inhibition of either VEGF (with soluble VEGF receptor 1 [sFlt1]) or fibroblast growth factor (FGF) (with a dominant=negative form of FGF receptor 1 [FGF-R1DN]), respectively, signaling reduced adventitial thickening induced by VEGF and PR39 to the level seen with mechanical arterial injury alone. However, neither inhibitor was effective in preventing neointimal thickening after mechanical injury when administered in the absence of angiogenic growth factor. CONCLUSIONS: Our findings indicate that adventitial angiogenesis stimulates intimal thickening but does not initiate it.
Angiogenesis Inducing Agents↗
Fibroblast growth factor 2 endocytosis in endothelial cells proceed via syndecan-4-dependent activation of Rac1 and a Cdc42-dependent macropinocytic pathway.
Full activity of fibroblast growth factors (FGFs) requires their internalization in addition to the interaction with cell surface receptors. Recent studies have suggested that the transmembrane proteoglycan syndecan-4 functions as a FGF2 receptor. In this study we investigated the molecular basis of syndecan endocytosis and its role in FGF2 internalization in endothelial cells. We found that syndecan-4 uptake, induced either by treatment with FGF2 or by antibody clustering, requires the integrity of plasma membrane lipid rafts for its initiation, occurs in a non-clathrin-, non-dynamin-dependent manner and involves Rac1, which is activated by syndecan-4 clustering. FGF2 was internalized in a complex with syndecan-4 in 70 kDa dextran-containing endocytic vesicles. FGF2 and syndecan-4 but not dextran endocytosis were blocked by the dominant negative Rac1 while amiloride and the dominant-negative Cdc42 blocked internalization of dextran in addition to FGF2 and syndecan-4. Taken together, these results demonstrate that FGF2 endocytosis requires syndecan-4 clustering-dependent activation of Rac1 and the intact CDC42-dependent macropinocytic pathway.
Animals↗
Post-market approval surveillance: a call for a more integrated and comprehensive approach.
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Clinical Trials as Topic↗
Medical device development: from prototype to regulatory approval.
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Academic Medical Centers↗
PR39 inhibits apoptosis in hypoxic endothelial cells: role of inhibitor apoptosis protein-2.
BACKGROUND: PR39 is a proline- and arginine-rich peptide implicated in wound healing and myocardial ischemia protection. To determine the potential mechanisms of PR39 in ischemia, we examined the role of PR39 in hypoxia-induced apoptosis in vascular endothelial cells. METHODS AND RESULTS: Hypoxia results in an increase of apoptosis in bovine aortic endothelial cells (BAECs), as determined by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labeling (TUNEL) analysis and caspase-3 activity. Hypoxia induced 66.2+/-2.7% TUNEL-positive cells, whereas in the presence of synthesized PR39 peptide, TUNEL-positive cells were reduced to 29.6+/-1.9% (P<0.05). After 24 hours of hypoxia, the addition of PR39 reduced caspase-3 activity to 3.17+/-0.47 pMol/min from 10.52+/-0.55 pMol/min in hypoxic BAECs. Moreover, PR39 increased inhibitor of apoptosis protein-2 (IAP-2) gene and protein expression by 3-fold in a time- and dose-dependent manner. The induction of IAP-2 by PR39 conferred an increase in IAP-2 gene transcription and IAP-2 mRNA stability. Furthermore, inhibiting IAP-2 with second mitochondria-derived activator of caspase (Smac) and with small interfering RNA targeting IAP-2 abrogated the ability of PR39 to reduce caspase-3 activity. CONCLUSIONS: We provide the first direct evidence for PR39 as an antiapoptotic factor in endothelial cells during hypoxia. These data suggest that PR39 inhibits hypoxia-induced apoptosis and decreases caspase-3 activity in endothelial cells through an increase of IAP-2 expression.
Amino Acid Sequence↗
Regulation of protein kinase B/Akt activity and Ser473 phosphorylation by protein kinase Calpha in endothelial cells.
Protein kinase Balpha (PKBalpha/Akt-1) is a key mediator of multiple signaling pathways involved in angiogenesis, cell proliferation and apoptosis among others. The unphosphorylated form of Akt-1 is virtually inactive and its full activation requires two phosphatidylinositol-3,4,5-triphosphate-dependent phosphorylation events, Thr308 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser473 by an undefined kinase that has been termed PDK2. Recent studies have suggested that the Ser473 kinase is a plasma membrane raft-associated kinase. In this study we show that protein kinase Calpha (PKCalpha) translocates to the membrane rafts in response to insulin growth factor-1 (IGF-1) stimulation. Overexpression of PKCalpha increases Ser473 phosphorylation and Akt-1 activity, while inhibition of its activity or expression decreases IGF-1-dependent activation of Akt-1. Furthermore, in vitro, in the presence of phospholipids and calcium, PKCalpha directly phosphorylates Akt-1 at the Ser473 site. We conclude, therefore, that PKCalpha regulates Akt-1 activity via Ser473 phosphorylation and may function as PDK2 in endothelial cells.
3-Phosphoinositide-Dependent Protein Kinases↗
Integrative signaling in angiogenesis.
Angiogenesis is a complex process requiring integration of multiple signals in order to achieve successful development of the new vasculature. While individual activities of numerous growth factors are well understood, the integration of their signaling at the cellular and tissue level is just beginning to be appreciated. This review focuses on these two process using vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) as examples.
Animals↗
Therapeutic angiogenesis for critical limb ischemia: design of the hepatocyte growth factor therapeutic angiogenesis clinical trial.
The objective of the HGF-STAT clinical trial is to determine whether perfusion can be improved by gene transfer with a plasmid DNA containing hepatocyte growth factor (HGF) in the affected limb of patients with unreconstructable critical limb ischemia (CLI). CLI results in a high rate of limb loss and impaired quality of life. The current therapeutic strategies, including bypass surgery and percutaneous interventions, are only successful in treating a subset of patients. Therapeutic angiogenesis is an investigational method that seeks to favorably impact tissue perfusion in CLI. HGF-STAT is a double-blind, parallel-group, placebo-controlled, dose-response study in 100 patients with unreconstructable CLI. Eligible subjects will be randomized 1:1:1:1 to receive saline placebo or one of three dose/regimens of HGF plasmid DNA. The selection of outcome measures, including the primary endpoint, and changes in transcutaneous oxygen pressure (TcPO2) from baseline to 3 months will be discussed. In conclusion, this study will help to determine whether therapeutic angiogenesis with HGF is a viable option in the treatment of patients with CLI.
Adult↗
Evolving revascularization approaches for myocardial ischemia.
Stable angina pectoris secondary to ischemic heart disease is a common and disabling condition. Medical therapy aims to relieve symptoms, improve exercise capacity, and decrease cardiac events by reducing myocardial oxygen demand or improving coronary blood supply to the ischemic myocardium. If medical treatment is inadequate, invasive revascularization procedures to improve coronary perfusion are considered. Percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass graft (CABG) surgery are well-established and widely used myocardial revascularization techniques. Recent advances in PTCA have attempted to address the problem of restenosis, initially through the deployment of bare metal intracoronary stents and, more recently, with drug-eluting stents. Developments in CABG have focused on reducing the invasiveness of the procedure and minimizing the incidence of serious complications. Refinements include the use of mechanical stabilizers, endoscopic harvesting of conduit vessels, robotic telemanipulation systems, and fully automated anastomotic devices. Surgical laser transmyocardial revascularization and therapeutic angiogenesis represent newer approaches to coronary revascularization. Therapeutic angiogenesis aims to deliver an angiogenic growth factor or cytokine to the myocardium to stimulate collateral blood vessel growth throughout the ischemic tissue. The angiogenic factor may be administered as a recombinant protein or as a transgene within a plasmid or gene-transfer vector. Ongoing angiogenic gene therapy clinical trials are evaluating which factors, vectors, and delivery techniques hold the greatest promise for management of patients with chronic stable angina.
Angiogenesis Inducing Agents↗
Proline- and arginine-rich peptides constitute a novel class of allosteric inhibitors of proteasome activity.
Substrate-specific inhibition of the proteasome has been unachievable despite great interest in proteasome inhibitors as drugs. Recent studies demonstrated that PR39, a natural proline- and arginine-rich antibacterial peptide, stimulates angiogenesis and inhibits inflammatory responses by specifically blocking degradation of IkappaBalpha and HIF-1alpha by the proteasome. However, molecular events involved in the PR39-proteasome interaction have not been elucidated. Here we show that PR39 is a noncompetitive and reversible inhibitor of the proteasome function. This effect is achieved by a unique allosteric mechanism allowing for specific inhibition of degradation of selected proteins without affecting total proteasome-dependent proteolysis. Atomic force microscopy (AFM) studies demonstrate that 20S and 26S proteasomes treated with PR39 or its derivatives exhibit serious perturbations in their structure and their normal allosteric movements. These effects are universal for proteasomes from yeast to human. The shortest functional sequence derived from PR39 still showing the allosteric inhibitory effect consists of eleven NH(2)-terminal residues containing essential three NH(2)-terminal arginines. The noncompetitive and reversible in vitro action of PR39 and its truncated derivatives is matched by the ability of the peptides to induce angiogenesis in vivo. We postulate that PR39 changes conformational dynamics of the proteasomes by interactions with the noncatalytic subunit alpha7 in a way that prevents the enzyme from cleaving the substrates of unique structural constraints.
Allosteric Site↗
Probucol prevents early coronary heart disease and death in the high-density lipoprotein receptor SR-BI/apolipoprotein E double knockout mouse.
Mice with homozygous null mutations in the high-density lipoprotein receptor SR-BI (scavenger receptor class B, type I) and apolipoprotein E genes fed a low-fat diet exhibit a constellation of pathologies shared with human atherosclerotic coronary heart disease (CHD): hypercholesterolemia, occlusive coronary atherosclerosis, myocardial infarctions, cardiac dysfunction (heart enlargement, reduced systolic function and ejection fraction, and ECG abnormalities), and premature death (mean age 6 weeks). They also exhibit a block in RBC maturation and abnormally high plasma unesterified-to-total cholesterol ratio (0.8) with associated abnormal lipoprotein morphology (lamellar/vesicular and stacked discoidal particles reminiscent of those in lecithin/cholesterol acyltransferase deficiency and cholestasis). Treatment with the lipid-lowering, antiatherosclerosis, and antioxidation drug probucol extended life to as long as 60 weeks (mean 36 weeks), and at 5-6 weeks of age, virtually completely reversed the cardiac and most RBC pathologies and corrected the unesterified to total cholesterol ratio (0.3) and associated distinctive abnormal lipoprotein morphologies. Manipulation of the timing of administration and withdrawal of probucol could control the onset of death and suggested that critical pathological changes usually occurred in untreated double knockout mice between approximately 3 (weaning) and 5 weeks of age and that probucol delayed heart failure even after development of substantial CHD. The ability of probucol treatment to modulate pathophysiology in the double knockout mice enhances the potential of this murine system for analysis of the pathophysiology of CHD and preclinical testing of new approaches for the prevention and treatment of cardiovascular disease.
Age Factors↗
Copper chelation represses the vascular response to injury.
The induction of an acute inflammatory response followed by the release of polypeptide cytokines and growth factors from peripheral blood monocytes has been implicated in mediating the response to vascular injury. Because the Cu2+-binding proteins IL-1alpha and fibroblast growth factor 1 are exported into the extracellular compartment in a stress-dependent manner by using intracellular Cu2+ to facilitate the formation of S100A13 heterotetrameric complexes and these signal peptideless polypeptides have been implicated as regulators of vascular injury in vivo, we examined the ability of Cu2+ chelation to repress neointimal thickening in response to injury. We observed that the oral administration of the Cu2+ chelator tetrathiomolybdate was able to reduce neointimal thickening after balloon injury in the rat. Interestingly, although immunohistochemical analysis of control neointimal sections exhibited prominent staining for MAC1, IL-1alpha, S100A13, and the acidic phospholipid phosphatidylserine, similar sections obtained from tetrathiomolybdate-treated animals did not. Further, adenoviral gene transfer of the IL-1 receptor antagonist during vascular injury also significantly reduced the area of neointimal thickening. Our data suggest that intracellular copper may be involved in mediating the response to injury in vivo by its ability to regulate the stress-induced release of IL-1alpha by using the nonclassical export mechanism employed by human peripheral blood mononuclear cells in vitro.
Adenoviridae↗
Spatial heterogeneity in VEGF-induced vasodilation: VEGF dilates microvessels but not epicardial and systemic arteries and veins.
This study was designed to investigate the site of vascular endothelial growth factor (VEGF)-induced vasodilation in the systemic and coronary vasculature. Intracoronary infusion of VEGF in Yorkshire pigs resulted in a significant drop in the mean arterial blood pressure, with a decline in the left ventricular left end-diastolic pressure, and no change in the heart rate. Coronary blood flow increase after intracoronary infusion of 10 mg VEGF (2.63 +/- 0.49x) was comparable to that seen after 40 mg of intracoronary adenosine (2.5 +/- 0.53x, p = 0.67) and was significantly higher then after 200 mg of intracoronary nitroglycerine (1.9 +/- 0.12x, p = 0.0005). At the same time, intracoronary VEGF did not result in a significant increase in coronary cross-sectional area determined using intravascular ultrasound. In vitro, VEGF produced dose-dependent relaxation of myocardial and systemic arterioles and venules (arterioles: 60-100 mm and venules: 120-200 mm in internal diameter) that was partially inhibited by L-NNA, but had no effect on epicardial coronary arteries, systemic arteries, or veins. Both VEGF receptors (flt-1 and flk-1) were identified on endothelial cells of epicardial arteries and veins. We conclude that this spatial heterogeneity of VEGF vasomotor effects cannot be explained by the absence VEGF receptors and suggests differential patterns of signal transduction in the vascular tree.
Adenosine↗