Gershon Golomb

VEGFR-1–Selective VEGF Homologue PlGF Is Arteriogenic: Evidence for a Monocyte-Mediated Mechanism

Abstract— Two signaling receptors for vascular endothelial growth factor (VEGF) in the vasculature are known with not yet well-understood roles in collateral vessel growth (arteriogenesis). In this study, we examined the involvement of the two VEGF receptors in arteriogenesis. Therefore, we used the VEGF homologue placenta growth factor (PlGF), which only binds to VEGFR-1 and VEGF-E, which only recognizes VEGFR-2. These peptides were locally infused over 7 days after ligation of the femoral artery in the rabbit. Evaluation of collateral growth by determining collateral conductance and angiographic scores demonstrated that the VEGFR-1–specific PlGF contributed significantly more to arteriogenesis than the VEGFR-2 specific VEGF-E. The combination of VEGF-E and PlGF did not exceed the effect of PlGF alone, indicating that cooperation of the two VEGF receptors in endothelial cell signaling is not required for arteriogenesis. In an in vitro model of angiogenesis, VEGF and VEGF-E were comparably active, whereas PlGF displayed no activity when given alone and did not further increase the effects of VEGF or VEGF-E. However, PlGF was as potent as VEGF when monocyte activation was assessed by monitoring integrin surface expression. In addition, accumulation of activated monocytes/macrophages in the periphery of collateral vessels in PlGF-treated animals was observed. Furthermore, in monocyte-depleted animals, the ability of PlGF to enhance collateral growth in the rabbit model and to rescue impaired arteriogenesis in PlGF gene–deficient mice was abrogated. Together, these data indicate that the arteriogenic activity observed with the VEGFR-1–specific PlGF is caused by its monocyte-activating properties.

Macrophage Depletion by Clodronate-Containing Liposomes Reduces Neointimal Formation After Balloon Injury in Rats and Rabbits

Background—Inflammation is critical to vascular repair after mechanical injury. Excessive inflammation enhances neointimal formation and restenosis. We examined whether transient systemic inactivation of macrophages at the time of vascular intervention could attenuate the degree of expected restenosis. Methods and Results—Liposomal clodronate (LC) inhibited the growth of cultured macrophages but had no effect on endothelial or smooth muscle cells and suppressed neointimal hyperplasia in hypercholesterolemic rabbits and rats after intravenous administration of LC, with no adverse effects. LC treatment reduced the number of blood monocytes and decreased macrophage infiltration in the injured arteries as well as smooth muscle cell proliferation, interleukin-1&bgr; transcription, and production and matrix metalloproteinase-2 activity. Conclusions—Macrophages play a pivotal role in vascular repair after mechanical arterial injury. Systemic inactivation and depletion of monocytes and macrophages by LC reduce neointimal hyperplasia and restenosis.

A new double emulsion solvent diffusion technique for encapsulating hydrophilic molecules in PLGA nanoparticles

The commonly utilized techniques for encapsulating hydrophilic molecules in NP suffer from low encapsulation efficiency because of the drug rapid partitioning to the external aqueous phase. We hypothesized that combining the double emulsion system with a partially water-soluble organic solvent, could result in better encapsulation yield of hydrophilic molecules in nano-sized NP, and the utilization of both biocompatible surfactants and solvents. As a model drug we used alendronate, a hydrophilic low MW bisphosphonate. The new NP preparation technique, double emulsion solvent diffusion (DES-D), resulted in improved formulation characteristics including smaller size, lower size distribution, higher encapsulation yield, and more biocompatible ingredients in comparison to classical methods. The utilization of partially water-miscible organic solvent (ethyl acetate) enabled rapid diffusion through the aqueous phase forming smaller NP. In addition, the formulated alendronate NP exhibited profound inhibition of raw 264 macrophages, depletion of rabbits circulating monocytes, and inhibition of restenosis in the rat model. It is concluded that the new technique is advantageous in terms of smaller size, lower size distribution, higher encapsulation yield, and more biocompatible ingredients, with unaltered bioactivity.

Spatiotemporal controlled delivery of nanoparticles to injured vasculature.

There are a number of challenges associated with designing nanoparticles for medical applications. We define two challenges here: (i) conventional targeting against up-regulated cell surface antigens is limited by heterogeneity in expression, and (ii) previous studies suggest that the optimal size of nanoparticles designed for systemic delivery is approximately 50–150 nm, yet this size range confers a high surface area-to-volume ratio, which results in fast diffusive drug release. Here, we achieve spatial control by biopanning a phage library to discover materials that target abundant vascular antigens exposed in disease. Next, we achieve temporal control by designing 60-nm hybrid nanoparticles with a lipid shell interface surrounding a polymer core, which is loaded with slow-eluting conjugates of paclitaxel for controlled ester hydrolysis and drug release over approximately 12 days. The nanoparticles inhibited human aortic smooth muscle cell proliferation in vitro and showed greater in vivo vascular retention during percutaneous angioplasty over nontargeted controls. This nanoparticle technology may potentially be used toward the treatment of injured vasculature, a clinical problem of primary importance.

PDGF-Receptor Tyrosine Kinase Blocker AG1295 Selectively Attenuates Smooth Muscle Cell Growth In Vitro and Reduces Neointimal Formation After Balloon Angioplasty in Swine

BACKGROUND Signaling through protein tyrosine kinases (PTKs) is a major contributor to the transmission of mitogenic stimuli to the interior of the cell and nucleus. The present study was designed to determine the effect of the tyrphostin AG1295, a selective blocker of PDGF-receptor PTK, on the growth of porcine and human smooth muscle cells (SMCs) in culture, on the outgrowth kinetics of SMCs from porcine and human arterial explants, and on neointimal formation after balloon injury in pigs. METHODS AND RESULTS SMCs for culture were obtained from porcine abdominal aortas, human internal mammary arteries, and endarterectomy tissue from a single human carotid artery. Addition of AG1295 to SMCs before PDGF stimulation completely inhibited PDGF-beta-receptor tyrosine phosphorylation without affecting the level of PDGF-beta-receptor. AG1295 resulted in a selective, reversible inhibition of SMC proliferation in culture (76%) with only mild (13.5%) inhibition of endothelial cell proliferation. The number of SMCs accumulating around explants of porcine carotid arteries and human endarterectomy specimens 12, 15, 19, 22, and 24 days after plating was reduced by 82% to 92% in AG1295-treated compared with nontreated specimens, and initiation of SMC outgrowth was markedly delayed. The numbers of cells accumulated 10 days after initiation of outgrowth were significantly lower in treated versus control explants. Local intravascular delivery of AG1295-impregnated polylactic acid-based nanoparticles (130+/-25 nm) to the site of balloon injury to porcine femoral arteries resulted in significant reductions in intima/media area ratio and luminal cross-sectional area narrowing by neointima compared with contralateral control arteries to which empty nanoparticles were applied (0.15+/-0.07 versus 0.09+/-0.03, P=.046 and 20+/-4% versus 10+/-4%, P=.0009, n=6 for both). CONCLUSIONS The tyrphostin AG1295, a selective blocker of PDGF-receptor kinase, exerts a marked inhibitory effect on the activation, migration, and proliferation of porcine and human SMCs in vitro and an approximately 50% inhibitory effect on neointimal formation after balloon injury in porcine femoral arteries when delivered via biodegradable nanoparticles. Further studies appear to be warranted to evaluate the applicability of this novel approach to the interventional setting.

Strategies for Treating Arterial Restenosis Using Polymeric Controlled Release Implants

Coronary artery obstruction is currently being treated with a number of invasive approaches involving catheter based angioplasty procedures. These have included most recently balloon angioplasty combined with expansion of obstructed coronary arteries using balloon expandable stainless steel stents. However, angioplasty itself, especially with stenting, leads to an accelerated reobstruction process, known as restenosis. Research reported in this paper has investigated an approach to preventing restenosis using controlled release drug-polymer implants for local inhibition of the pathophysiologic events of restenosis. Model therapeutic compounds were chosen including aspirin, as an antiplatelet agent, hirulog, as an antithrombin, and colchicine as an antiproliferative. Controlled release polymer matrices were successfully formulated and characterized. Retention of anticoagulant activity for the peptide, hirulog, was demonstrated in vitro. These polymers are suitable for investigations in periadventitial implants and animal models of restenosis. Eventually, controlled release strategies for preventing restenosis will involve integrating of ideal agents including gene therapy, with stents and related devices in order to develop a drug delivery systems approach.

Transplacental effects of bisphosphonates on fetal skeletal ossification and mineralization in rats

Bisphosphonates are clinically used mainly to reduce bone resorption. We studied the transplacental effects of two bisphosphonates on the fetal skeleton in rats. Pregnant rats were treated during days 11-20 of pregnancy with daily subcutaneous injections of 0.1 mg/kg of alendronate or a newly synthesized bisphosphonate, VS-b6. This period of pregnancy was chosen because the active development of bones from mesenchyme through cartilaginous models occurs during that time. Histological examination of midlongitudinal sections of the 21-day-old fetuses showed an increase in the amount of diaphyseal bone trabeculae with slight shortening of the diaphysis in the experimental fetuses, in comparison to controls. Computerized histomorphometric studies similarly showed an increase in the amount of diaphyseal bone trabeculae with a concomitant decrease in bone marrow volume, but no change in cartilage volume. In addition, chemical analysis of the fetal bones showed an increase in calcium content in the treated fetuses. 14C-alendronate was shown to pass through the rat placenta and accumulate in the fetuses, most probably in their bones. This is presumed because bisphosphonates are known to accumulate in bone, being stored there for long periods of time. It is important, in light of our results, to give careful consideration to the treatment of women with bisphosphonates at childbearing age, whenever this is needed.

Delivery of serotonin to the brain by monocytes following phagocytosis of liposomes

Many drugs are not able to enter the brain due to the presence of the blood-brain barrier (BBB) and therefore cannot be used in the treatment of diseases of the brain. Since it is now known that the brain is under immunological surveillance, we hypothesized that phagocytic cells of the innate immune system, mainly neutrophils and monocytes, can be exploited as transporters of drugs to the brain. To target circulating mononuclear phagocytic cells, negatively-charged nano-sized liposomes were formulated encapsulating serotonin, a BBB impermeable neurological drug. Brain uptake, biodistribution, and the mechanism of brain transport were examined in vitro and in rats and rabbits by utilizing double-radiolabeled (3)H (in the membrane) and (14)C-serotonin (in the core), and liposomes with fluorescent markers (membrane and core). The brain uptake of liposomal serotonin was significantly higher (0.138%+/-0.034 and 0.097%+/-0.011, vs. 0.068%+/-0.02 and 0.057%+/-0.01, 4 h and 24 h after IV administration in rats, serotonin liposomes and in solution, respectively). The same brain uptake of both empty and serotonin liposomes, the co-localization in the brain of both markers, and the unchanged ratio of (3)H:(14)C suggest that intact liposomes entered the brain. Since treatment of animals by liposomal alendronate resulted with inhibition of monocytes but not of neutrophils, and with no brain delivery, it is suggested that monocytes are the main transporters of liposomes to the brain.

In vivo prevention of arterial restenosis with paclitaxel-encapsulated targeted lipid–polymeric nanoparticles

Following recent successes with percutaneous coronary intervention (PCI) for treating coronary artery disease (CAD), many challenges remain. In particular, mechanical injury from the procedure results in extensive endothelial denudation, exposing the underlying collagen IV-rich basal lamina, which promotes both intravascular thrombosis and smooth muscle proliferation. Previously, we reported the engineering of collagen IV-targeting nanoparticles (NPs) and demonstrated their preferential localization to sites of arterial injury. Here, we develop a systemically administered, targeted NP system to deliver an antiproliferative agent to injured vasculature. Approximately 60-nm lipid–polymeric NPs were surface functionalized with collagen IV-targeting peptides and loaded with paclitaxel. In safety studies, the targeted NPs showed no signs of toxicity and a ≥3.5-fold improved maximum tolerated dose versus paclitaxel. In efficacy studies using a rat carotid injury model, paclitaxel (0.3 mg/kg or 1 mg/kg) was i.v. administered postprocedure on days 0 and 5. The targeted NP group resulted in lower neointima-to-media (N/M) scores at 2 wk versus control groups of saline, paclitaxel, or nontargeted NPs. Compared with sham-injury groups, an ∼50% reduction in arterial stenosis was observed with targeted NP treatment. The combination of improved tolerability, sustained release, and vascular targeting could potentially provide a safe and efficacious option in the management of CAD.

Liposomal Alendronate Inhibits Systemic Innate Immunity and Reduces In-Stent Neointimal Hyperplasia in Rabbits

Background—Innate immunity is of major importance in vascular repair. The present study evaluated whether systemic and transient depletion of monocytes and macrophages with liposome-encapsulated bisphosphonates inhibits experimental in-stent neointimal formation. Methods and Results—Rabbits fed on a hypercholesterolemic diet underwent bilateral iliac artery balloon denudation and stent deployment. Liposomal alendronate (3 or 6 mg/kg) was given concurrently with stenting. Monocyte counts were reduced by >90% 24 to 48 hours after a single injection of liposomal alendronate, returning to basal levels at 6 days. This treatment significantly reduced intimal area at 28 days, from 3.88±0.93 to 2.08±0.58 and 2.16±0.62 mm2. Lumen area was increased from 2.87±0.44 to 3.57±0.65 and 3.45±0.58 mm2, and arterial stenosis was reduced from 58±11% to 37±8% and 38±7% in controls, rabbits treated with 3 mg/kg, and rabbits treated with 6 mg/kg, respectively (mean±SD, n=8 rabbits/group, P <0.01 for all 3 parameters). No drug-related adverse effects were observed. Reduction in neointimal formation was associated with reduced arterial macrophage infiltration and proliferation at 6 days and with an equal reduction in intimal macrophage and smooth muscle cell content at 28 days after injury. Conversely, drug regimens ineffective in reducing monocyte levels did not inhibit neointimal formation. Conclusions—Systemic transient depletion of monocytes and macrophages, by a single liposomal bisphosphonates injection concurrent with injury, reduces in-stent neointimal formation and arterial stenosis in hypercholesterolemic rabbits.