Cerebrovascular super-resolution 4D Flow MRI - Sequential combination of resolution enhancement by deep learning and physics-informed image processing to non-invasively quantify intracranial velocity, flow, and relative pressure.

The development of cerebrovascular disease is tightly coupled to regional changes in intracranial flow and relative pressure. Image-based assessment using phase contrast magnetic resonance imaging has particular promise for non-invasive full-field mapping of cerebrovascular hemodynamics. However, estimations are complicated by the narrow and tortuous intracranial vasculature, with accurate image-based quantification directly dependent on sufficient spatial resolution. Further, extended scan times are required for high-resolution acquisitions, and most clinical acquisitions are performed at comparably low resolution (>1 mm) where biases have been observed with regard to the quantification of both flow and relative pressure. The aim of our study was to develop an approach for quantitative intracranial super-resolution 4D Flow MRI, with effective resolution enhancement achieved by a dedicated deep residual network, and with accurate quantification of functional relative pressures achieved by subsequent physics-informed image processing. To achieve this, our two-step approach was trained and validated in a patient-specific in-silico cohort, showing good accuracy in estimating velocity (relative error: 15.0 ± 0.1%, mean absolute error (MAE): 0.07 ± 0.06 m/s, and cosine similarity: 0.99 ± 0.06 at peak velocity) and flow (relative error: 6.6 ± 4.7%, root mean square error (RMSE): 0.56 mL/s at peak flow), and with the coupled physics-informed image analysis allowing for maintained recovery of functional relative pressure throughout the circle of Willis (relative error: 11.0 ± 7.3%, RMSE: 0.3 ± 0.2 mmHg). Furthermore, the quantitative super-resolution approach is applied to an in-vivo volunteer cohort, effectively generating intracranial flow images at <0.5 mm resolution and showing reduced low-resolution bias in relative pressure estimation. Our work thus presents a promising two-step approach to non-invasively quantify cerebrovascular hemodynamics, being applicable to dedicated clinical cohorts in the future.

Numerical Simulation of Stent Angioplasty with Predilation: An Investigation into Lesion Constitutive Representation and Calcification Influence.

It is acceptable clinical practice to predilate a severely occluded vessel to allow better positioning of endovascular stents, and while the impact of this intervention has been examined for aggregate response in animals there has been no means to examine whether there are specific vessels that might benefit. Finite element methods offer the singular ability to explore the mechanical response of arteries with specific pathologic alterations in mechanics to stenting and predilation. We examined varying representations of atherosclerotic tissue including homogeneous and heterogeneous dispersion of calcified particles, and elastic, pseudo-elastic, and elastic-plastic constitutive representations of bulk atherosclerotic tissue. The constitutive representations of the bulk atherosclerotic tissue were derived from experimental test data and highlight the importance of accounting for testing mode of loading. The impact of arterial predilation is presented and, in particular, its effect on intimal predicted damage, atherosclerotic tissue von Mises and maximum principal stresses, and luminal deformation was dependent on the type of constitutive representation of diseased tissue, particularly in the presence of calcifications.

Diffusion-limited binding explains binary dose response for local arterial and tumour drug delivery.

BACKGROUND: Local drug delivery has transformed medicine, yet it remains unclear how drug efficacy depends on physicochemical properties and delivery kinetics. Most therapies seek to prolong release, yet recent studies demonstrate sustained clinical benefit following local bolus endovascular delivery. OBJECTIVES: The purpose of the current study was to examine interplay between drug dose, diffusion and binding in determining tissue penetration and effect. METHODS: We introduce a quantitative framework that balances dose, saturable binding and diffusion, and measured the specific binding parameters of drugs to target tissues. RESULTS: Model reduction techniques augmented by numerical simulations revealed that impact of saturable binding on drug transport and retention is determined by the magnitude of a binding potential, B(p), ratio of binding capacity to product of equilibrium dissociation constant and accessible tissue volume fraction. At low B(p) (< 1), drugs are predominantly free and transport scales linearly with concentration. At high B(p) (> 40), drug transport exhibits threshold dependence on applied surface concentration. CONCLUSIONS: In this paradigm, drugs and antibodies with large B(p) penetrate faster and deeper into tissues when presented at high concentrations. Threshold dependence of tissue transport on applied surface concentration of paclitaxel and rapamycin may explain threshold dose dependence of in vivo biological efficacy of these drugs.

Phospholipase C-delta extends intercellular signalling range and responses to injury-released growth factors in non-excitable cells.

OBJECTIVES: Intercellular communication in non-excitable cells is restricted to a limited range close to the signal source. Here, we have examined whether modification of the intracellular microenvironment could prolong the spatial proposition of signal generation and could increase cell proliferation. MATERIAL AND METHODS: Mathematical models and experimental studies of endothelial repair after controlled mechanical injury were used. The models predict the diffusion range of injury-released growth factors and identify important parameters involved in a signalling regenerative mode. Transfected human umbilical vein endothelial cells (HUVECs) were used to validate model results, by examining intercellular calcium signalling range, cell proliferation and wound healing rate. RESULTS: The models predict that growth factors have a limited capacity of extracellular diffusion and that intercellular signals are specially sensitive to cell phospholipase C-delta (PLCdelta) levels. As basal PLCdelta levels are increased by transfection, a significantly increased intercellular calcium range, enhanced cell proliferation, and faster wound healing rate were observed. CONCLUSION: Our in silico and in vitro studies demonstrated that non-excitable endothelial cells respond to stimuli in a complex manner, in which intercellular communication is controlled by physicochemical properties of the stimulus and by the cell microenvironment. Such findings may have profound implications for our understanding of the tight nature of autocrine cell growth control, compensation to stress states and response to altered microenvironment, under pathological conditions.

Innate immunity has a dual effect on vascular healing: suppression and aggravation of neointimal formation and remodeling post-endotoxin challenge.

BACKGROUND: Inflammation is important to vascular repair following injury, modulating neointimal proliferation and remodeling. Previously, we have shown that a low-intensity inflammatory response aggravates neointimal formation following balloon and stent injury. The present study examined whether modulation of the extent and timing of nonspecific inflammation mediates the local vascular response in an additive unidirectional or rather a bidirectional fashion. METHODS AND RESULTS: Rabbits subjected to denudation and balloon injury of the iliac artery were treated with low (1 microg/kg) or high (100 microg/kg) doses of bacterial endotoxin (LPS) immediately after injury, or with early high-dose LPS administered 3 days prior to injury (preconditioning). Neointimal formation at 28 days was significantly increased in the low-dose group (0.537+/-0.059 mm(2)) as compared with controls (0.3+/-0.03 mm(2)). High-dose LPS did not significantly affect neointimal formation while early high dose significantly reduced neointima (0.296+/-0.033 and 0.194+/-0.025 mm(2), respectively, n=12-14/group). Arterial wall and systemically circulating interleukin-1 beta levels, and monocyte CD14 activation correlated with neointimal formation. Vascular remodeling was accelerated in animals treated with low- or high-dose LPS while not affected in the preconditioned group. Remodeling index inversely correlated with arterial matrix metalloproteinase-2 levels 6 days after injury. CONCLUSIONS: The extent and timing of nonspecific inflammation that is concurrent with vascular injury can determine different and opposite vascular repair patterns.

Tissue engineering of endothelial cells and the immune response.

BACKGROUND: While tissue engineering offers promise for organ and tissue transplantation, it can also be used to examine transplant and immune biology. Endothelial cells engrafted within 3-dimensional matrices create stable units that produce all of the factors of a functional quiescent endothelium. Perivascular implantation of tissue engineered endothelial cell constructs provides long-term control of vascular repair after injury. This control is established without restoration of the natural luminal:mural endothelium, and most intriguingly, without engendering host allo- and xenogeneic immune responses. We examined how endothelial immunogenicity is controlled by interaction with 3-dimensional matrices. MATERIALS AND METHODS: Human aortic endothelial cells (HAE) were either grown to confluence on polystyrene tissue culture plates or within 3-dimensional collagen-based matrices. Major histocompatibility complex (MHC) class II, integrin, interferon (IFN)-gamma receptor expression, and signaling were analyzed via confocal microscopy, flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), and microarray. Splenocyte proliferation was assayed by thymidine incorporation. RESULTS: Despite similar expression levels of IFN-gamma receptors, matrix-embedded HAE elicited far less STAT-1 phosphorylation upon IFN-gamma stimulation, and expressed 2-fold less MHC II than HAE grown to confluence on culture plates (P < .001). This effect correlated with reduced expression of integrin alpha(v) and beta(3) (P < .002), and muted proliferation of porcine splenocytes (P < .001). CONCLUSIONS: Matrix architecture is critical for modulation of endothelial immunogenicity. Embedding HAE within a physiologic 3-dimensional environment affects activity of intracellular signaling pathways, MHC II expression, and subsequent activation of immune cells. These findings might offer novel insights into our understanding of endothelial-mediated diseases and might enhance our ability to leverage the potential for cell-based therapies.

The total quasi-steady-state approximation is valid for reversible enzyme kinetics.

The Briggs-Haldane approximation of the irreversible Michaelis-Menten scheme of enzyme kinetics is cited in virtually every biochemistry textbook and is widely considered the classic example of a quasi-steady-state approximation. Though of similar importance, the reversible Michaelis-Menten scheme is not as well characterized. This is a serious limitation since even enzymatic reactions that go to completion may be reversible. The current work derives a total quasi-steady-state approximation (tQSSA) for the reversible Michaelis-Menten and delineates its validity domain. The tQSSA allows the derivation of uniformly valid approximations for the limit of low enzyme concentrations, ET<

Carrier proteins determine local pharmacokinetics and arterial distribution of paclitaxel.

The growing use of local drug delivery to vascular tissues has increased interest in hydrophobic compounds. The binding of these drugs to serum proteins raises their levels in solution, but hinders their distribution through tissues. Inside the arterial interstitium, viscous and steric forces and binding interactions impede drug motion. As such, this might be the ideal scenario for increasing the amount of drug delivered to, and residence time within, arterial tissues. We quantified carrier-mediated transport for paclitaxel, a model hydrophobic agent with potential use in proliferative vascular diseases, by determining, in the presence or absence of carrier proteins, the maximum concentration of drug in aqueous solution, the diffusivity in free solution, and the diffusivity in arterial tissues. Whereas solubility of paclitaxel was raised 8.1-, 21-, and 57-fold by physiologic levels of alpha(1)-acid glycoproteins, bovine serum albumin, and calf serum over that in protein-free solution, diffusivity of paclitaxel in free solution was reduced by 41, 49, and 74%, respectively. When paclitaxel mixed in these solutions was applied to arteries both in vitro and in vivo, drug was more abundant at the tissue interface, but protein carriers tended to retain drug in the lumen. Once within the tissue, these proteins did not affect the rate at which drug traverses the tissue because this hydrophobic drug interacted with the abundant fixed proteins and binding sites. The protein binding properties of hydrophobic compounds allow for beneficial effects on transvascular transport, deposition, and distribution, and may enable prolonged effect and rationally guide local and systemic strategies for their administration.

Oral heparin prevents neointimal hyperplasia after arterial injury: inhibitory potential depends on type of vascular injury.

BACKGROUND: In animal models, heparin delivered as a continuous intravenous infusion or via frequent (BID) subcutaneous dosing inhibits neointimal hyperplasia after balloon injury or stent implantation. However, human trials of subcutaneous heparin after percutaneous intervention have proven ineffective against restenosis. It may be that these failures are due to unfavorable heparin pharmacokinetics. Recently, the drug delivery agent sodium N-[8-(2-hydroxybenzoyl)amino] caprylate (SNAC) has been found to facilitate the gastric absorption of heparin. METHODS AND RESULTS: To investigate the effects of orally delivered heparin on neointimal hyperplasia after varying forms of arterial injury, 57 New Zealand White rabbits underwent iliac artery balloon dilatation. In half of the rabbits, endovascular stents were implanted and heparin was delivered through a variety of methods. Arteries were harvested at 14 days. Neointimal area was assessed with computer-aided morphometry. After balloon injury, both intravenous (0.3 mg/kg per hour) and oral heparin (90 mg/kg BID) effectively inhibited neointimal hyperplasia (0.11+/-0.02 and 0.09+/-0.07 mm(2), respectively, versus 0.16+/-0.06 mm(2) in control; P<0.05). After stent implantation, intravenous administration of heparin (0.3 mg/kg per hour) effectively inhibited neointimal growth (0.35+/-0.05 mm(2) versus 0.51+/-0.09 mm(2) in control; P<0.05), but oral heparin at 90 mg/kg BID and 180 mg/kg BID (0.48+/-0.04 and 0.49+/-0.08 mm(2), respectively; P=NS versus control) did not. A dose of 120 mg/kg TID, however, was effective (0.40+/-0.10 mm(2); P<0.05 versus control). CONCLUSIONS: These data suggest that oral heparin may be an effective therapy against restenosis after percutaneous intervention. Stented arteries required higher and more frequent dosing for efficacy. These data suggest that differences in the type of vascular injury must be considered in the design of drug delivery.

Physiological transport forces govern drug distribution for stent-based delivery.

BACKGROUND: The first compounds considered for stent-based delivery, such as heparin, were chosen on the basis of promising tissue culture and animal experiments, and yet they have failed to stop restenosis clinically. More recent compounds, such as paclitaxel, are of a different sort, being hydrophobic in nature, and their effects after local release seem far more profound. This dichotomy raises the question of whether drugs that have an effect when released from a stent do so because of differences in biology or differences in physicochemical properties and targeting. METHODS AND RESULTS: We applied continuum pharmacokinetics to examine the effects of transport forces and device geometry on the distribution of stent-delivered hydrophilic and hydrophobic drugs. We found that stent-based delivery invariably leads to large concentration gradients, with drug concentrations ranging from nil to several times the mean tissue concentration over a few micrometers. Concentration variations were a function of the Peclet number (Pe), the ratio of convective to diffusive forces. Although hydrophobic drugs exhibited greater variability than hydrophilic drugs, they achieved higher mean concentrations and remained closer to the intima. Inhomogeneous strut placement influenced hydrophilic drugs more negatively than hydrophobic drugs, dramatically affecting local concentrations without changing mean concentrations. CONCLUSIONS: Because local concentrations and gradients are inextricably linked to biological effect, our results provide a potential explanation for the variable success of stent-based delivery. We conclude that mere proximity of delivery devices to tissues does not ensure adequate targeting, because physiological transport forces cause local concentrations to deviate significantly from mean concentrations.

Endothelial implants provide long-term control of vascular repair in a porcine model of arterial injury.

Cell culture and animal data support the role of endothelial cells and endothelial-based compounds in regulating vascular repair after injury. We describe a long-term study in pigs in which the biological and immunological responses to endothelial cell implants were investigated 3 months after angioplasty, approximately 2 months after the implants have degraded. Confluent porcine or bovine endothelial cells grown in polymer matrices were implanted adjacent to 28 injured porcine carotid arteries. Porcine and bovine endothelial cell implants significantly reduced experimental restenosis compared to control by 56 and 31%, respectively. Host humoral responses were investigated by detection of an increase in serum antibodies that bind to the bovine or porcine cell strains used for implantation. A significant increase in titer of circulating antibodies to the bovine cells was observed after 4 days in all animals implanted with xenogeneic cells. Detected antibodies returned to presurgery levels after Day 40. No significant increase in titer of antibodies to the porcine cells was observed during the time course of the experiment in animals implanted with porcine endothelial cells. No implanted cells, Gelfoam, or focal inflammatory reaction could be detected histologically at any of the implant sites at 90 days. These data suggest that tissue-engineered endothelial cell implants may provide long-term control of vascular repair after injury, rather than simply delaying lesion formation and that allogeneic implants are able to provide a greater benefit than xenogeneic implants.

The dorsal fin engine of the seahorse (Hippocampus sp.).

The muscles, fin ray joints, and supporting structures underlying the dorsal fin are described for two seahorse species: Hippocampus zosterae and Hippocampus erectus. A fan-shaped array of cartilaginous bones, the pterigiophores, form the internal supporting structure of the dorsal fin. Each pterigiophore is composed of a proximal radial that extends from a vertebra to the dorsal side of the animal, where it fuses to a middle radial. The middle radials fuse with each other to form a dorsal ridge upon which sit the spheroidal distal radials. Each distal radial articulates with a fin ray on its dorsal side and is attached to the dorsal ridge on its ventral side by a material that has been histologically identified as elastic cartilage. Together these connections form a two-axis joint that permits elevation, depression, and inclination of the ray. Each fin ray is actuated by two bilateral pairs of muscles, an anterior pair of inclinators, and a posterior pair of depressors. The anteriormost fin ray is actuated by three bilateral pair of muscles, the inclinators, the depressors, and a pair of elevator muscles that are positioned anterior to the inclinators. Preliminary examinations of the ray joints of the pectoral and anal fins of adult H. zostera and the pectoral fins of newborn H. erectus revealed structures similar to that seen in the dorsal fins. To further explore the structure and function of the dorsal fin gross dissections and simple functional tests were performed on H. erectus and H. barbouri and behavioral observations were made of all three species plus Hippocampus kuda.

Gold-coated NIR stents in porcine coronary arteries.

BACKGROUND: As endovascular stents are altered to add functionality, eg, by adding radiopaque coatings, biocompatibility may suffer. METHODS AND RESULTS: We examined the vascular response in porcine coronary arteries to stainless steel gold-coated NIR stents (7-cell, Medinol, Inc). Stents, 9 and 16 mm in length, were left bare or coated with a 7-microm layer of gold. Physical and material effects were examined in four different gold-coated stent types, two at each length that either had the coating applied to the standard strut, ie, gold coated thicker than controls, or had the coating applied to thinned struts, ie, gold coated of the same thickness as control struts. Simple gold coating exacerbated intimal hyperplastic and inflammatory reactions over 28 days, but postplating thermal processing smoothed the coating surface and negated the adverse tissue response to gold. The relative amounts of base steel and gold coating and their resistances to expansion and collapse determined the extent of stent recoil. CONCLUSIONS: Gold coatings enhance the radiopacity of steel stents, but not without effects on vascular repair. Material effects predominate and can be abrogated by heating coated stents to alter surface finish and material purity. Clinical results may suffer unless consideration is given to material and physical effects of gold.

Neointimal thickening after stent delivery of paclitaxel: change in composition and arrest of growth over six months.

OBJECTIVES: The purpose of this study was to determine long-term effects of stent-based paclitaxel delivery on amount, rate and composition of neointimal thickening after stent implantation. BACKGROUND: Paclitaxel prevents vascular smooth muscle cell proliferation and migration in vitro and in vivo. These actions, coupled with low solubility, make it a viable candidate for modulating vascular responses to injury and prolonged effects after local delivery. We asked whether local delivery of paclitaxel for a period of weeks from a stent coated with a bioerodible polymer could produce a sustained reduction in neointimal hyperplasia for up to six months after stenting. METHODS: Stainless steel stents were implanted in the iliac arteries of rabbits after endothelial denudation. Stents were uncoated or coated with a thin layer of poly(lactide-co-sigma-caprolactone) copolymer alone or containing paclitaxel, 200 microg. RESULTS: Paclitaxel release in vitro followed first-order kinetics for two months. Tissue responses were examined 7, 28, 56 or 180 days after implantation. Paclitaxel reduced intimal and medial cell proliferation three-fold seven days after stenting and virtually eliminated later intimal thickening. Six months after stenting, long after drug release and polymer degradation were likely complete, neointimal area was two-fold lower in paclitaxel-releasing stents. Tissue responses in paclitaxel-treated vessels included incomplete healing, few smooth muscle cells, late persistence of macrophages and dense fibrin with little collagen. CONCLUSIONS: Poly(lactide-co-sigma-caprolactone) copolymer-coated stents permit sustained paclitaxel delivery in a manner that virtually abolishes neointimal hyperplasia for months after stent implantation, long after likely completion of drug delivery and polymer degradation.

Neutrophil, not macrophage, infiltration precedes neointimal thickening in balloon-injured arteries.

Macrophages are abundant after stent-induced arterial injury. Inhibition of macrophage recruitment blocks neointimal growth in this model. In contrast, after superficial injury from balloon endothelial denudation, macrophages are sparse. However, many anti-inflammatory therapies remain effective against neointimal growth after balloon injury. To investigate further the role of leukocytes after injury, 41 New Zealand White rabbits underwent iliac artery balloon denudation. In 18, subcutaneous pumps were placed to deliver intravenous heparin (0.3 mg/kg per hour). Arteries were harvested at 6 hours and at 3, 7, and 14 days. In 8 animals, either M1/70 (a monoclonal antibody [mAb] against adhesion molecule Mac-1) or a nonspecific IgG was given (5 mg/kg IV bolus and then 1 mg/kg SC QOD), and arteries were harvested at 6 hours and 3 days. Computer-aided morphometry was performed as was immunohistochemistry to assess smooth muscle cell (SMC) proliferation (bromodeoxyuridine-positive cells), neutrophil content (RPN357, mAb against rabbit neutrophil/thymocyte), and macrophage content (RAM-11, mAb against rabbit macrophage). Heparin inhibited neointimal growth at 7 and 14 days (64% and 32.5% reduction, respectively; P:<0.05). Neutrophils were observed in the media early after balloon injury, and heparin and M1/70 inhibited this infiltration (82% and 83% reduction, respectively; P:<0.05 each) with a coincident inhibition of medial SMC proliferation at 3 days (49% and 84% reduction, respectively; P:<0.05 each). Macrophages were absent at all time points. Neutrophil, but not macrophage, infiltration occurs early after endothelial denudation. Inhibition of this process is associated with a reduction in medial SMC proliferation. These data suggest a central role for neutrophils in restenosis and help to explain prior reports of an inhibitory effect of anti-inflammatory therapies on neointimal growth after balloon injury.

Perivascular graft heparin delivery using biodegradable polymer wraps.

Heparin remains the gold-standard inhibitor of the processes involved in the vascular response to injury. Though this compound has profound and wide-reaching effects on vascular cells in culture and animal models, its clinical utility has been questionable at best. It is clear that the mode of heparin delivery is critical to its potential and it may well be that routine forms of administration are insufficient to observe benefit given the heparin's short half-life and complex pharmacokinetics. When ingested orally, heparin is degraded to inactive oligomer fragments while systemic administration is complicated by the need for continuous infusion and the potential for uncontrolled hemorrhage. Thus alternative heparin delivery systems have been proposed to maximize regional effects while limiting systemic toxicity. Yet, as heparin is such a potent antithrombotic compound and since existing local delivery systems lack the ability to precisely regulate release kinetics, even site-specific therapy is prone to bleeding. We now describe the design and development of a novel biodegradable system for the perivascular delivery of heparin to the blood vessel wall with well-defined release kinetics. This system consists of heparin-encapsulated poly(DL lactide-co-glycolide) (pLGA) microspheres sequestered in an alginate gel. Controlled release of heparin from this heterogeneous system could be obtained over a period of 25 days in vitro. The experimental variables affecting heparin release from these matrices were investigated. Gel permeation chromatography (GPC) and scanning electron microscopy (SEM) were used to monitor the degradation process and found to correlate well with the release kinetics. Heparin-releasing gels inhibited growth of bovine vascular smooth muscle cells in tissue culture in a dose-dependent manner. Moreover, gel release controlled vascular injury in denuding and interposition vascular graft animal models of disease even when uncontrolled bleeding was evident with standard matrix-type release. This system may therefore provide an effective means of examining the effects of various compounds in the control of smooth muscle cell proliferation in accelerated arteriopathies and also shed light on the biologic nature of these processes.

Local drug delivery: an emerging approach in the treatment of restenosis.

Very limited success has been demonstrated with systemic pharmacological treatment to reduce the incidence of restenosis following angioplasty in patients. The lack of success of many of the pharmacotherapeutic agents in reducing the restenosis rates post-angioplasty and following stent implementation is believed to arise from inadequate concentrations of the agents at the lesion site. This has led to the development of various local delivery devices that would ideally deliver and retain adequate amounts of drug to the vessel wall for sufficient periods of time to ensure a therapeutic effect without inducing further injury or compromising blood flow. Local dosing would avoid systemic toxicity, and the use of modified balloon catheters or coated stents might enable percutaneous approaches.

Endothelial cell delivery for cardiovascular therapy.

Obstructive atherosclerotic vascular disease stands as one of the greatest public health threats in the world. While a number of therapies have been developed to combat vascular disease, endothelial cell delivery has emerged as a distinct therapeutic modality. In this article, we will review the anatomy of the normal blood vessel and the biology of the intact endothelium, focusing upon its centrality in vascular biology and control over the components of the vascular response to injury so as to understand better the motivation for a cell-based form of therapy. Our discussion of cell delivery for cardiovascular therapy will be divided into surgical and interventional approaches. We will briefly recount the development of artificial grafts for surgical vascular bypass before turning our attention towards endothelial cell seeded vascular grafts, in which endothelial cells effectively provide local delivery of endogenous endothelial secretory products to maintain prosthetic integrity after surgical implantation. New techniques in tissue and genetic engineering of vascular grafts and whole blood vessels will be presented. Methods for percutaneous interventions will be examined as well. We will evaluate results of endoluminal endothelial cell seeding for treatment of restenosis and gene therapy approaches to enhance endogenous re-endothelialization. Finally, we will examine some innovations in endothelial cell delivery that may lead to the development of endothelial cell implants as a novel therapy for controlling proliferative vascular arteriopathy.

Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia.

Perlecan, a heparan sulfate proteoglycan, has been suggested to be critical for regulation of vascular repair. We generated clones of endothelial cells expressing an antisense vector targeting domain III of perlecan. Transfected cells produced significantly less perlecan than parent cells and showed a reduced ability to inhibit the binding and mitogenic activity of fibroblast growth factor-2 in vascular smooth muscle cells. Endothelial cells were seeded onto three-dimensional polymeric matrices and implanted adjacent to porcine carotid arteries subjected to deep injury. Although the parent endothelial cells prevented occlusive thrombosis, perlecan-deficient cells were completely ineffective. The ability of endothelial cells to inhibit intimal hyperplasia, however, was abrogated only in part by perlecan suppression. The differential regulation by perlecan of these different aspects of vascular repair may explain why control of clinical clot formation does not lead to full control of intimal hyperplasia. Thus the use of genetically modified tissue-engineered cells provides a new approach for dissecting the role of specific factors within the complex environment of the blood vessel wall.