Angiogenic endothelial cell invasion into fibrin is stimulated by proliferating smooth muscle cells.

These studies aimed to determine the effect of smooth muscle cells (SMCs) on angiogenic behavior of endothelial cells (ECs) within fibrin hydrogels, an extracellular matrix (ECM) commonly used in tissue engineering. We developed a 3-D, fibrin-based co-culture assay of angiogenesis consisting of aggregates of SMCs with ECs seeded onto the aggregates' surface. Using digital fluorescence micrography, EC matrix invasion was quantified by average length of sprouts (ALS) and density of sprout formation (DSF). We demonstrated that ECs and SMCs co-invade into the ECM in close proximity to one another. ECs that were co-cultured with SMCs demonstrated increased invasion compared to ECs that were cultured alone at all time points. At Day 19, the ALS of ECs in co-culture was 327+/-58µm versus 70+/-11µm of ECs cultured alone (p=.01). The DSF of co-cultured ECs was also significantly greater than that of ECs cultured alone (p=.007 on Day 19). This appeared to be a function of both increased EC invasion as well as improved persistence of EC sprout networks. At 7days, ECs in co-culture with proliferation-inhibited SMCs previously treated with Mitomycin-C (MMC) demonstrated significantly attenuated sprouting compared to ECs co-cultured with SMCs that were untreated with MMC (82+/-14µm versus 205+/-32µm; p<.05). In assays in which multiple co-culture aggregates were cultured within a single hydrogel, we observed directional invasion of sprouts preferentially towards the other aggregates within the hydrogel. In co-culture assays without early EC/SMC contact, the ALS of ECs cultured in the presence of SMCs was significantly greater than those cultured in the absence of SMCs by Day 3 (320+/-21µm versus 187+/-16µm; p<.005). We conclude that SMCs augment EC matrix invasion into 3-D fibrin hydrogels, at least in part resulting from SMC proliferative and invasive activities. Directed invasion between co-culture aggregates and augmented angiogenesis in the absence of early contact suggests a paracrine mechanism for the observed results.

Paclitaxel inhibits ureteral smooth muscle cell proliferation and collagen production in the absence of cell toxicity.

PURPOSE: Urinary tract stricture results from excess collagen deposition at an injured area. Paclitaxel (Sigma-Aldrich®) prevents coronary artery restenosis by inhibiting vascular smooth muscle cell proliferation and collagen production. We evaluated the effects of paclitaxel on ureteral smooth muscle cell proliferation and collagen production. MATERIALS AND METHODS: Three phases of experiments were done in canine smooth muscle cells. In phase 1 we used proliferation assay to study smooth muscle cells exposed to various concentrations of paclitaxel during 7 days. Phase 2 consisted of 6-day enzyme-linked immunosorbent assay to detect the total amount of type III collagen produced by smooth muscle cells exposed to paclitaxel. In phase 3 we assessed smooth muscle cell membrane damage using a lactate dehydrogenase cytotoxicity assay in which cells were exposed to escalating paclitaxel concentrations for 14 days. RESULTS: Proliferation studies showed that 10 and 100 nM paclitaxel significantly inhibited ureteral smooth muscle cell proliferation. Enzyme-linked immunosorbent assay revealed significantly decreased type III collagen production at 100 nM. Cytotoxicity testing showed that 1 to 100 nM paclitaxel did not harm smooth muscle cells. CONCLUSIONS: Paclitaxel effectively inhibits canine ureteral smooth muscle cell proliferation and collagen production without toxicity to smooth muscle cells at concentrations up to 100 nM. These results may ultimately translate into new methods of preventing and treating urinary stricture disease.

Fibroblast growth factor-1 (FGF-1) loaded microbeads enhance local capillary neovascularization.

BACKGROUND: Growth of new blood vessels (neovascularization) occurs naturally in the body, but the slow rate of the process may not be sufficient for survival of engineered tissues and transplanted cells, such as pancreatic islets. For transplanted islets, it is crucial that the transplantation site has sufficient vasculature to support the needs of the islets. Therefore, the specific aim of this research was quantify the effect of FGF-1 incorporation into alginate microbeads on neovascularization of such capsules in an in vivo rat transplant model. MATERIALS AND METHODS: Microbeads loaded with FGF-1 or control beads (beads without FGF-1) were implanted in the rat omental pouch model. Animals were sacrificed 7 d post-implantation. RESULTS: Microbeads loaded with FGF-1 stimulated a significant increase in vascular density compared with control rats implanted with control beads. CONCLUSIONS: These results indicate that alginate microbeads loaded with FGF-1 enhance local neovascularization around implanted microbeads. These data provide a compelling impetus for experimental pursuit of FGF-loaded alginate microcapsules for vascularization of transplanted islets.

Construction and characterization of a thrombin-resistant designer FGF-based collagen binding domain angiogen.

Humans demonstrate limited spontaneous endothelialization of prosthetic bypass grafts. However the local application of growth factors to prosthetic grafts or to injured blood vessels can provide an immediate effect on endothelialization. Novel chimeric proteins combining potent angiogens with extracellular matrix binding domains may localize to exposed matrices and provide sustained activity to promote endothelial regeneration after vascular interventions. We have ligated a thrombin-resistant mutant of fibroblast growth factor (FGF)-1 (R136K) with a collagen binding domain (CBD) in order to direct this growth factor to sites of exposed vascular collagen or selected bioengineered scaffolds. While FGF-1 and R136K are readily attracted to a variety of matrix proteins, R136K-CBD demonstrated selective and avid binding to collagen approximately 4x that of FGF-1 or R136K alone (P<0.05). The molecular stability of R136K-CBD was superior to FGF-1 and R136K. Its chemotactic activity was superior to R136K and FGF-1 (11+/-1% vs. 6+/-2% and 4+/-1%; P<0.01). Its angiogenic activity was similar to R136K and significantly greater than control by day 2 (P<0.01). After day 3, FGF-1-treated endothelial cell's (EC) sprouts had regressed back to levels insignificant compared to the control group (P=0.17), while both R136K and R136K-CBD continued to demonstrate greater sprout lengthening as compared to control (P<0.0002). The mitogenic activity of all growth factors was greater than control groups (20% PBS); in all comparisons (P<0.0001). This dual functioning angiogen provides proof of concept for the application of designer angiogens to matrix binding proteins to intelligently promote endothelial regeneration of selected matrices.

Growing a living blood vessel: insights for the second hundred years.

Cardiovascular disease continues to be the leading cause of death worldwide, and the prevalence of cardiovascular disease has reached epidemic proportions worldwide. Not surprisingly this has led to an increasing number of vascular procedures annually. Unfortunately, the success of these procedures over time continues to limit their long-term effects. Biomedical engineering approaches to improve upon current prosthetic grafts, developing new prosthetic grafts, and creating tissue engineered blood vessels for clinical application offer hope of improving the durability of vascular interventions and improving patients' treatment for cardiovascular disease.

Therapeutic neovascularization: contributions from bioengineering.

A number of pathological entities and surgical interventions could benefit from therapeutic stimulation of new blood vessel formation. Although strategies designed for promoting neovascularization have shown promise in preclinical models, translation to human application has met with limited success when angiogenesis is used as the single therapeutic mechanism. While clinical protocols continue to be optimized, a number of exciting new approaches are being developed. Bioengineering has played an important role in the progress of many of these innovative new strategies. In this review, we present a general outline of therapeutic neovascularization, with an emphasis on investigations using engineering principles to address this vexing clinical problem. In addition, we identify some limitations and suggest areas for future research.

Angiogenic effect of fibroblast growth factor-1 and vascular endothelial growth factor and their synergism in a novel in vitro quantitative fibrin-based 3-dimensional angiogenesis system.

BACKGROUND: We have developed an in vitro 3-dimensional angiogenesis system in which the length, distribution, and ultrastructure of induced capillary sprouts were analyzed in response to concentration ranges of fibroblast growth factor (FGF)-1 and vascular endothelial growth factor (VEGF) and synergistic activity quantitated. METHODS: Bovine aorta endothelial cell aggregates were embedded in fibrin gel (FG) supported by a nylon mesh ring. The formed disks were cultured in 24-well plates in assay media. The test growth factors FGF-1, VEGF, or both (0 to 100 ng/mL) with 100 KIU/mL aprotinin were added to the media. The disks (n = 8/group) were digitally photographed and capillary sprouts quantitated. Assay disks were then fixed and sectioned for morphology. RESULTS: In aprotinin-stabilized FG, aggregated ECs invaded FG radially, forming sprouts and capillary networks. Neovessel lumens surrounded by ECs were confirmed on hematoxylin and eosin and transmission electron microscopy and by formation of cell junctions by transmission electron microscopy. The angiogenic effects of FGF-1 and VEGF were dose-dependent in the range from 1 to 100 ng/mL. Significant activity of FGF-1 started at 1 ng/mL and of VEGF at 2 ng/mL. The greatest effect was at the highest concentration (100 ng/mL) for both cytokines. The combination of 10 ng/mL of each FGF-1 and VEGF induced a significantly greater effect than the additive effects of FGF-1 (10 ng/mL) or VEGF (10 ng/mL) alone when analyzed with SAS system for mixed model (P <.0001), and that also exceeded the effects by 20 ng/mL of either FGF-1 or VEGF. CONCLUSIONS: A 3-dimensional capillary network can be induced in aprotinin-stabilized FG using FGF-1 or VEGF with synergism between the 2 angiogens.

The effect of intraoperative duplex on the management of postoperative stroke.

BACKGROUND: Stroke after carotid endarterectomy (CEA) may be a result of intraoperative ischemia, embolism, or thrombosis at the operative site. Intraoperative duplex should eliminate the occurrence of a severe internal carotid artery (ICA) thrombosis and, thus, negate the benefit of reoperation. This article will detail the results of our evolving treatment algorithm for immediate versus delayed post-CEA neurologic deficit (ND). METHODS: We studied patients who had an ND after CEA from 1988 to 2000. Results. Thirty-two patients (3.2%) had a post-CEA ND (26 related stroke or transient ischemic attack, 6 other); 31 had a satisfactory intraoperative duplex post-CEA, 1 was not tested. Fifteen patients awoke from operation with a related deficit, 5 of whom were re-explored and all had a patent ICA. One patient without lateralizing signs who was not re-explored had extensive thrombosis at postmortem. The remaining 9 all had a duplex-proven patent ICA. Ten patients had a lucid interval before their related ND. Six patients were re-explored and all had thrombosed ICAs; 5 of the 6 improved postthrombectomy. Four patients were not re-explored for various reasons; a carotid thrombosis was not later diagnosed in any of these patients. CONCLUSIONS: Intraoperative and postoperative duplex has modified our treatment of post-CEA stroke. No longer are all patients re-explored. Patients with a normal intraoperative duplex who awaken with an immediate stroke do not usually have occlusive thrombus and routine re-exploration does not benefit these patients. Patients who have an ND develop after a lucid period may have a thrombosed ICA despite a normal intraoperative duplex, and unless there is a timely normal duplex, re-exploration is recommended and appears to benefit these patients.

Silyl-heparin adsorption improves the in vivo thromboresistance of carbon-coated polytetrafluoroethylene vascular grafts.

BACKGROUND: Expanded polytetrafluoroethylene (ePTFE) remains the most commonly utilized synthetic graft material for infrainguinal arterial reconstruction. However, patency rates of ePTFE bypass grafts are inferior to those observed with autogenous vein grafts. Modification of the luminal surface of ePTFE grafts such as coating with carbon or heparin, may prevent early graft failures and improve overall patency rates. We now report our results with a silyl-heparin adsorbed carbon-coated ePTFE graft. METHODS: Silyl-heparin was adsorbed onto carbon-coated ePTFE vascular grafts (Bard Peripheral Vascular, Tempe, Arizona), which were then evaluated for patency and platelet deposition acutely (2 hours after implantation) and 7 days after graft implantation in mongrel dogs. Dogs underwent bilateral aortoiliac grafting where one heparin adsorbed carbon-coated graft and one carbon-coated graft (control) were placed on either (alternating) side. Platelet deposition was determined by injection of autologous (111)Indium radiolabeled platelets followed by a 2-hour circulation period prior to explantation of grafts. Heparin activity of the silyl-heparin grafts (at preimplantation and explantation) was determined using an antithrombin-III based thrombin binding assay. RESULTS: Graft patency was 100% for both heparin coated (5 of 5) grafts and control (5 of 5) grafts in the acute group of dogs. In the 7-day group, patency was 87.5% for heparin coated (7 of 8) grafts and 50% for control (4 of 8) grafts (P = 0.28, Fisher's exact test). Radiolabeled platelet studies revealed a significantly lower deposition of platelets on heparin coated grafts compared with control grafts in the acute group (17.3 +/- 13.5 versus 35.2 +/- 17.9 counts per minute, per cm(2) per million platelets, mean +/- SEM; n = 5, P <0.05, paired Student t test). In the 7-day group of dogs with bilaterally patent grafts (4 of 8), a trend toward a lower deposition of platelets on heparin coated grafts compared with control grafts was observed (1.55 +/- 0.409 versus 2.14 +/- 1.13 counts per minute, per cm(2) per million platelets, mean +/- SEM; n = 4, P = 0.52, paired Student t test). Eight percent of the preimplantation heparin activity remained on the explanted silyl-heparin grafts after 2 hours and only 2% after 7 days. CONCLUSIONS: Silyl-heparin adsorption onto carbon-coated ePTFE vascular grafts resulted in improved acute thromboresistance in a canine bilateral aortoiliac model. Ongoing laboratory efforts are aimed at improving the silyl-heparin retention on vascular grafts. This graft may prove to be useful in the clinical setting.

Heparin-independent mitogenicity in an endothelial and smooth muscle cell chimeric growth factor (S130K-HBGAM).

BACKGROUND: Through site-directed mutagenesis we have created a favorable fibroblast growth factor-1 (FGF-1) mutant (S130K) and linked it to a heparin-binding growth-associated molecule (HBGAM) to form the chimera S130K-HBGAM creating a heparin-independent, endothelial cell (EC)-specific mitogen. METHODS: The proliferative responses of primary canine carotid artery smooth muscle cells (SMC) and jugular vein EC to FGF-1, S130K, or S130K-HBGAM, with and without heparin (5 U/mL), was quantitated by measuring tritiated thymidine uptake over 24 hours and expressing the results as percent of positive control (20% fetal bovine serum [FBS]) for group comparison. RESULTS: Unlike FGF-1, both S130K and S130K-HBGAM are heparin-independent mitogens for EC and SMC. S130K-HBGAM was equivalent to FGF-1 with heparin at 6 nmol/L. S130K-HBGAM did not demonstrate relative EC specificity in this assay. CONCLUSIONS: At higher concentrations, S130K-HBGAM is a potent, heparin-independent EC and SMC mitogen. Co-culture assays and in vivo delivery models may demonstrate EC specificity not identified in this single cell type proliferation assay.

Vascular grafts.

Autogenous vein is the conduit of choice in the surgical creation of bypasses of small-to-medium-caliber vessels in patients with peripheral occlusive arterial disease and will remain so for the near future. The success rate of bypasses using conduits of diameters greater than 6 mm has been excellent, whereas the majority of bypasses using smaller conduits fail within 5 years. In addition, due to a steep increase in rates of diabetes and decreasing cardiovascular mortality rates, increasing challenges are presented by this population. These facts have motivated much of the research in the cardiovascular arena over the past four decades, with improved techniques and new materials. Strategies to improve outcomes include the use of alternative materials including autologous, nonautologous and prosthetic grafts, utilizing different methods for their harvesting and preservation; tissue engineering, using either polymer- or biological-based scaffolds for cell seeding; endovascular methodologies; and gene therapy. This report presents an overview of the several options currently available in the management of patients with peripheral arterial occlusive disease, as well as the ongoing research directed towards the creation of an artificial engineered vessel, discussing experimental work in which endothelial cells have been seeded on different scaffolds and finally the potential application of gene therapy in the field of vascular reconstruction.

Comparison of standard carotid endarterectomy with Dacron patch angioplasty versus eversion carotid endarterectomy during a 4-year period.

Currently, the two primary approaches to carotid endarterectomy for extracranial carotid stenosis are carotid endarterectomy with patch angioplasty and eversion carotid endarterectomy. In a retrospective study over a 4-year period from 1998 to 2002, we had an opportunity to compare the two approaches as two surgeons utilized carotid endarterectomy with Dacron patch angioplasty and two other surgeons utilized eversion carotid endarterectomy. During the 4-year period, 189 carotid endarterectomies were performed, 125 with Dacron patch angioplasty (CE-P) and 64 with eversion (EE) endarterectomy. There were no significant differences in age of the patients, operative indication, or associated risk factors between the two groups. Perioperative outcome measurement in the CE-P versus EE included stroke or transient ischemic attack, 1.6 per cent versus 1.56 per cent, cranial nerve injury, 2.4 per cent versus 3.13 per cent; death, 0.8 per cent versus 0 per cent; need for operative conversion or revision, 2.4 per cent versus 7.81 per cent, respectively. Only the need for operative conversion or revision reached significant difference (P < 0.05), although the need decreased to 4 per cent for the last 50 EE cases. Recurrent stenosis of 50 per cent to 79 per cent was 4.88 per cent versus 3.13 per cent and >80 per cent was 0.81 per cent versus 0 per cent in the CE-P versus EE group over a follow up of 16.3 months and 17.0 months, respectively. We conclude that both CE-P and EE are equally efficacious operative approaches to extracranial carotid occlusive disease.

Analyzer-based phase-contrast x-ray imaging of carotid plaque microstructure.

BACKGROUND: Plaque vulnerability depends, in part, on composition. Imaging techniques are needed that can aid the prediction of plaque stability. High-contrast images of soft-tissue structure have been obtained with x-ray phase-contrast (PC) imaging. This research investigates multiple image radiography (MIR), an x-ray PC imaging technique, for evaluation of human carotid artery plaques. METHODS: Carotid plaques were imaged with ultrasound and subsequently excised and formalin fixed. MIR imaging was performed. By using synchrotron radiation, conventional radiographs were acquired for comparison. Image texture measures were computed for soft-tissue regions of the plaques. RESULTS: Ultrasound evaluation identified plaques as homogeneous without calcifications. MIR images revealed complex heterogeneous structure with multiple microcalcifications consistent with histology, and possessed more image texture in specific regions than conventional radiographs (P < .05). MIR refraction images allowed imaging of the geometric structure of tissue interfaces within the plaques, while scatter images contained more texture in soft-tissue regions than absorption or refraction images. CONCLUSIONS: X-ray PC imaging better depicts plaque soft-tissue heterogeneity than ultrasound or conventional radiographs. MIR imaging technique should be investigated further as a viable imaging technique to identify high-risk plaques.

Dynamic quantitative visualization of single cell alignment and migration and matrix remodeling in 3-D collagen hydrogels under mechanical force.

We developed a live imaging system enabling dynamic visualization of single cell alignment induced by external mechanical force in a 3-D collagen matrix. The alignment dynamics and migration of smooth muscle cells (SMCs) were studied by time lapse differential interference contrast and/or phase contrast microscopy. Fluorescent and reflection confocal microcopy were used to study the SMC morphology and the microscale collagen matrix remodeling induced by SMCs. A custom developed program was used to quantify the cell migration and matrix remodeling. Our system enables cell concentration-independent alignment eliminating cell-to-cell interference and enables dynamic cell tracking, high magnification observation and rapid cell alignment accomplished in a few hours compared to days in traditional models. We observed that cells sense and response to the mechanical signal before cell spreading. Under mechanical stretch the migration directionality index of SMCs is 46.3% more than those cells without external stretch; the dynamic direction of cell protrusion is aligned to that of the mechanical force; SMCs showed directional matrix remodeling and the alignment index calculated from the matrix in front of cell protrusions is about 3 fold of that adjacent to cell bodies. Our results indicate that the mechanism of cell alignment is directional cell protrusion. Mechano-sensing, directionality in cell protrusion dynamics, cell migration and matrix remodeling are highly integrated. Our system provides a platform for studying the role of mechanical force on the cell matrix interactions and thus finds strategies to optimize selected properties of engineered tissues.

Three-dimensional 10% cyclic strain reduces bovine aortic endothelial cell angiogenic sprout length and augments tubulogenesis in tubular fibrin hydrogels.

The development of a functional microvasculature is critical to the long-term survival of implanted tissue-engineered constructs. Dynamic culture conditions have been shown to significantly modulate phenotypic characteristics and stimulate proliferation of cells within hydrogel-based tissue engineered blood vessels. Although prior work has described the effects uniaxial or equibiaxial mechanical stimulation has on endothelial cells, no work has outlined effects of three-dimensional mechanical stimulation on endothelial cells within tubular vessel analogues. We demonstrate here that 7 days of 10% cyclic volumetric distension has a deleterious effect on the average length and density of angiogenic sprouts derived from pellets of bovine aortic endothelial cells. Although both groups demonstrated lumen formation, the sprouts grown under dynamic culture conditions typically had wider, less-branching sprout patterns. These results suggest that prolonged mechanical stimulation could represent a cue for angiogenic sprouts to preferentially develop larger lumens over cellular migration and subsequent sprout length.

Using a type 1 collagen-based system to understand cell-scaffold interactions and to deliver chimeric collagen-binding growth factors for vascular tissue engineering.

Vascular tissue engineering should provide more biocompatible and functional conduits than synthetic vascular grafts. Understanding cell-scaffold interactions and developing an efficient delivery system for growth factors and other biomolecules to control the signaling between the cells and the scaffold are fundamental issues in a wide range of tissue engineering research fields. Type 1 collagen is a natural scaffold extensively used in vascular tissue engineering and is a widely used vehicle in biomolecule delivery. In this article, we will discuss type 1 collagen as a vascular tissue engineering scaffold, describe strategies for elucidating the interaction between cells and type 1 collagen scaffolds using various imaging techniques, and summarize our work on the development of a chimeric collagen-binding growth factor-based local delivery system.

Molecular mediators of angiogenesis.

Angiogenesis, or the formation of new blood vessels from the preexisting vasculature, is a key component in numerous physiologic and pathologic responses and has broad impact in many medical and surgical specialties. In this review, we discuss the key cellular steps that lead to the neovascularization of tissues and highlight the main molecular mechanisms and mediators in this process. We include discussions on proteolytic enzymes, cell-matrix interactions, and pertinent cell signaling pathways and end with a survey of the mechanisms that lead to the stabilization and maturation of neovasculatures.