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.

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.

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.

Multilayered microcapsules for the sustained-release of angiogenic proteins from encapsulated cells.

BACKGROUND: Multilayered alginate microcapsules with a permselective poly-L-ornithine membrane can be used for the dual purpose of encapsulating cells in the inner core and sustained release of angiogenic proteins from the outer layer. The aim of this study was to examine the encapsulation and release of a novel chimeric form of fibroblast growth factor-1 (FGF-1) from the outer layer of alginate microcapsules. METHODS: Heparin-binding growth-associated molecule bound to FGF-1 (HB-GAM/FGF-1) was encapsulated in the outer layer of multilayered alginate microbeads constructed using varying alginate conditions. The encapsulation and release of the chimera was quantified. RESULTS: The outer layer was able to encapsulate and release HB-GAM/FGF-1 for up to 30 days. The outer layer made with 1% alginate of high mannuronic acid content provided the fastest release, while 1.25% high guluronic acid content alginate displayed the longest duration of release. CONCLUSIONS: The outer layer of multilayered alginate microbeads can be used for the encapsulation and long-term release of HB-GAM/FGF-1.

Characterization of the chemotactic and mitogenic response of SMCs to PDGF-BB and FGF-2 in fibrin hydrogels.

The delivery of growth factors to cellularize biocompatible scaffolds like fibrin is a commonly used strategy in tissue engineering. We characterized smooth muscle cells (SMC) proliferation and chemotaxis in response to PDGF-BB and FGF-2, alone and in combination, in 2D culture and in 3D fibrin hydrogels. While both growth factors induced an equipotent mitogenic response in 2D culture, only FGF-2 was significantly mitogenic for SMCs in 3D culture. Only PDGF-BB was significantly chemotactic in a modified Boyden chamber assay. In a 3D assay of matrix invasion, both growth factors induced an invasive response into the fibrin hydrogel in both proliferating and nonproliferating, mitomycin C (MMC) treated cells. The invasive response was less attenuated by the inhibition of proliferation in PDGF-BB stimulated cells compared with FGF-2 stimulated cells. We conclude that SMCs cultured in fibrin hydrogels have a more robust chemotactic response to PDGF-BB compared with FGF-2, and that the response to FGF-2 is more dependent on cell proliferation. Delivery of both growth factors together potentiates the chemotactic, but not mitogenic response to either growth factor alone.

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.

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.

Local delivery of a collagen-binding FGF-1 chimera to smooth muscle cells in collagen scaffolds for vascular tissue engineering.

We investigated the delivery of R136K-CBD (a collagen-binding mutant chimera of fibroblast growth factor-1) with a type I collagen scaffold as the delivery vehicle to smooth muscle cells (SMCs) for vascular tissue engineering. The binding affinity of R136K-CBD to 3-D collagen scaffolds was investigated both in the presence and absence of cells and/or salts. 2-D and 3-D visualization of delivery of R136K-CBD into SMCs were accomplished by combined fluorescent and reflection confocal microscopy. The mitogenic effect of collagen-immobilized R136K-CBD on SMCs in 3-D collagen was studied by Cyquant assay at different time intervals. In the group devoid of salt and cells, no detectable release of R136K-CBD into overlying culture media was found, compared with burst-and-continuous release of R136K and FGF-1 over a 14-day period in all other groups. The release rate of R136K-CBD was 1.7 and 1.6-fold less than R-136K and FGF-1 when media was supplemented with 2m salt (P<0.0001), and 2.6 and 2.5-fold less in cell-populated collagen hydrogels (P<0.0001), respectively. R136K-CBD showed essentially uniform binding to collagen and its distribution was dependent on that of the collagen scaffold. Internalization of R136K-CBD into SMCs was documented by confocal microscopy. 3-D local delivery of collagen-immobilized R136K-CBD increased the proliferation of SMCs in the collagen matrix to significantly greater levels and for a significantly greater duration than R136K or FGF-1, with 2.0 and 2.1-fold more mitogenicity than R136K and FGF-1 respectively (P<0.0001) at day 7. The results suggest that our collagen-binding fusion protein is an effective strategy for growth factor delivery for vascular tissue engineering.

The temporal and spatial dynamics of microscale collagen scaffold remodeling by smooth muscle cells.

Smooth muscle cells (SMCs) and collagen scaffolds are widely used in vascular tissue engineering but their interactions in remodeling at the microscale level remained unclear. We characterized microscale morphologic alterations of collagen remodeled by SMCs in six dimensions: three spatial, time, multichannel and multi-position dimensions. In live imaging assays, computer-assisted cell tracking showed locomotion characteristics of SMCs; reflection and fluorescent confocal microscopy and spatial reconstruction images of each time point showed detailed morphologic changes of collagen fibers and spatial collagen-SMC interactions. The density of the collagen around the SMCs was changed dynamically by the leading edges of the cells. The density of the collagen following 24h of cell-induced remodeling increased 51.61+/-9.73% compared to unremodeled collagen containing cells for 1h (P<0.0001, n=40) (NS vs. collagen without cells). Fast Fourier transform analysis showed that the collagen fibers' orientation changed from random (alignment index=0.047+/-0.029, n=40) after 1h into concordant with that of the SMCs (alignment index=0.379+/-0.098, P<0.0001, n=40) after 24h. Mosaic imaging extended the visual field from a single cell to a group of cells in one image without loss of optical resolution. Direct visualization of alignment of actin fibers and collagen fibers showed the molecular machinery of the process of scaffold remodeling. This is a new approach to better understanding the mechanism of scaffold remodeling and our techniques represent effective tools to investigate the interactions between cells and scaffold in detail at the microscale level.

Characterization of type I collagen gels modified by glycation.

Chronic exposure to reducing sugars due to diabetes, aging, and diet can permanently modify extracellular matrix (ECM) proteins. This non-enzymatic glycosylation, or glycation, can lead to the formation of advanced glycation end products (AGE) and crosslinking of the ECM. This study investigates the effects of glycation on the properties of type I collagen gels. Incubation with glucose-6-phopshate (G6P), a reducing sugar that exhibits similar but more rapid glycation than glucose, modified the biological and mechanical properties of collagen gels. Measures of AGE formation that correlate with increased complications in people with diabetes, including collagen autofluorescence, crosslinking, and resistance to proteolytic degradation, increased with G6P concentration. Rheology studies showed that AGE crosslinking increased the shear storage and loss moduli of type I collagen gels. Fibroblasts cultured on glycated collagen gels proliferated more rapidly than on unmodified gels, but glycated collagen decreased fibroblast invasion. These results show that incubation of type I collagen gels with G6P increases clinically relevant measures of AGE formation and that these changes altered cellular interactions. These gels could be used as in vitro models to study ECM changes that occur in diabetes and aging.

Biomimetic control of vascular smooth muscle cell morphology and phenotype for functional tissue-engineered small-diameter blood vessels.

Small-diameter blood vessel substitutes are urgently needed for patients requiring replacements of their coronary and below-the-knee vessels and for better arteriovenous dialysis shunts. Circulatory diseases, especially those arising from atherosclerosis, are the predominant cause of mortality and morbidity in the developed world. Current therapies include the use of autologous vessels or synthetic materials as vessel replacements. The limited availability of healthy vessels for use as bypass grafts and the failure of purely synthetic materials in small-diameter sites necessitate the development of a biological substitute. Tissue engineering is such an approach and has achieved promising results, but reconstruction of a functional vascular tunica media, with circumferentially oriented contractile smooth muscle cells (SMCs) and extracellular matrix, appropriate mechanical properties, and vasoactivity has yet to be demonstrated. This review focuses on strategies to effect the switch of SMC phenotype from synthetic to contractile, which is regarded as crucial for the engineering of a functional vascular media. The synthetic SMC phenotype is desired initially for cell proliferation and tissue remodeling, but the contractile phenotype is then necessary for sufficient vasoactivity and inhibition of neointima formation. The factors governing the switch to a more contractile phenotype with in vitro culture are reviewed.

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.

In vitro models of angiogenesis.

Neovascularization can be categorized into two general processes: vasculogenesis and angiogenesis. Angiogenesis is the formation of new capillaries from pre-existing vessels, requiring growth factor driven recruitment, migration, proliferation, and differentiation of endothelial cells (ECs). Complex cell-cell and cell-extracellular matrix (ECM) interactions contribute to this process, leading finally to a network of tube-like formations of endothelial cells supported by surrounding mural cells. The study of angiogenesis has broad clinical implications in the fields of peripheral and coronary vascular disease, oncology, hematology, wound healing, dermatology, and ophthalmology, among others. As such, novel, clinically relevant models of angiogenesis in vitro are crucial to the understanding of angiogenic processes. We highlight some of the advances made in the development of these models, and discuss the importance of incorporating the three-dimensional cell-matrix and EC-mural cell interactions into these in vitro assays of angiogenesis. This review also discusses our own 3-D angiogenesis assay and some of the in vitro results from our lab as they relate to therapeutic neovascularization and tissue engineering of vascular grafts.

Improving endothelial healing with novel chimeric mitogens.

BACKGROUND: Chimeric proteins may be used to direct cell-specific activity. Heparin-binding growth-associated molecule (HBGAM) binds to cell receptors that are relatively more robust on endothelial cells, and it may confer endothelial cell selectivity to potent angiogens such as fibroblast growth factor-1 (FGF-1). METHODS: By ligating fibroblast growth factor or its potent mutant, S130K, to HBGAM, we tested their effect on re-endothelialization after angioplasty injury by using a canine model. RESULTS: Both HBGAM/S130K- and HBGAM/FGF-1-treated arteries had increased neointimal mitotic index and re-endothelialization rates at 30 days compared with control arteries without inducing a significant increase in the neointimal thickness or the ratio of neointimal to medial thickness between treatment and control groups. CONCLUSION: HBGAM/S130K and HBGAM/FGF-1 facilitates endothelial healing without myointimal thickening after canine carotid artery balloon angioplasty injury. Application of these growth factors in fibrin glue may improve endothelialization clinically after angioplasty or endarterectomy.