The Interplay between Biomechanical and Biochemical Factors Regulates. Lumen Formation and Navigation of Endothelial Cell Sprouts. Amir Shamloo1, Sarah ...
Proceedings of the ASME 2010 Summer Bioengineering Conference SBC2010 June 16-19, 2010, Naples, Florida, USA
SBC2010-19495 The Interplay between Biomechanical and Biochemical Factors Regulates Lumen Formation and Navigation of Endothelial Cell Sprouts Amir Shamloo1, Sarah C. Heilshorn2 1
Mechanical Engineering, 2Materials Science and Engineering, Stanford University, Stanford, CA
Introduction: Angiogenesis is the process of forming new blood vessels that originate from pre-existing vessels. In early angiogenesis stages, endothelial cells (ECs) migrate from the lumen of developed blood vessels into the surrounding extracellular matrix (ECM). Through the coordinated actions of migration and proliferation, these ECs organize into tubular capillary-like structures called sprouts. In this study, 3D EC sprout formation was examined using a microfluidic device that enabled the separate and simultaneous tuning of biomechanical and biochemical stimuli (Fig. 1). While previous investigations have been performed on each of these factors individually1, 2, more recent studies have identified a critical interplay between the simultaneous effects of these two factors3. For example, we previously studied 2D EC chemotaxis in response to vascular endothelial growth factor (VEGF) gradients in the absence of biomechanical stimulation.4 In developing a model that enables precise specification of biochemical and biomechanical cues, we utilized a protocol that enables ECs to undergo a transition from the 2D to 3D culture environment mimicking angiogenic sprouting. Here we quantified the relative importance and combined consequences of discrete changes in matrix density, growth factor concentration, and growth factor gradient steepness during the stages of early sprout initiation, sprout elongation, sprout navigation, and lumen formation. Methods: Soft lithography was used to fabricate the microfluidic mold. Human dermal microvascular endothelial cells were cultured on the 2D surfaces of microcarrier dextran beads (d~170 mm). The EC-coated beads were mixed with a collagen-fibronectin gel precursor solution and injected into the microfluidic device cell culture chamber. Medium with and without VEGF was continuously injected into the source and sink channels, respectively, to form a stable VEGF concentration gradient across the cell culture chamber (Fig. 1). Matrix density was tailored by altering the collagen concentration within the gel while maintaining a constant fibronectin concentration. Oscillatory rheometry was used to characterize gel viscoelasticity. Results: Five collagen gel densities (0.3, 0.7, 1.2, 1.9 and 2.7 mg/ml) were selected to determine the effects of matrix biomechanics on the formation of EC capillarylike sprouts. These selected densities produce gels with plateau storage moduli, G’, ranging from 7-700 Pa. Within a VEGF gradient at low collagen densities (0.3 mg/ml), ECs quickly migrated into the matrix as single cells without forming cohesive sprout structures (Fig. 2A). At slightly increased collagen densities (0.7 mg/ml), tracks of multiple cells began to coordinate their
migration to form unstable sprout structures within 24 hours (Fig. 2B). In marked contrast, stable sprouts were formed at intermediate collagen densities after two to three days in culture (1.2 mg/ml, 1.9 mg/ml; Fig. 2C,D). At the highest tested collagen density (2.7 mg/ml), the cells assembled into clusters that formed thick protrusions on the bead and did not elongate into sprouts (Fig. 2E). The time required for new sprouts to assemble was increased with increasing matrix density, suggesting that the rate of new sprout formation is limited by the migration speed through the 3D matrix (Fig. 2F). We observed that sprout thickness increased and sprout length decreased with increasing matrix density (Fig. 2G). Stable sprout formation per bead was maximized at intermediate collagen densities (1.2 and 1.9 mg/ml; Fig. 2H). B
Figure 1. Microfluidic device schematic and gradient formation. A. The device is composed of source and sink channels connected to a cell culture chamber by microcapillaries. B. The cell culture chamber is loaded with a collagen gel (pink) containing endothelial cell-coated microcarrier beads.
Sprout navigation within these two intermediate collagen densities was characterized in response to three VEGF profiles including steeper and shallower VEGF gradients with identical absolute VEGF concentrations (0-50 ng/ml). To quantify navigation; sprout initiation point, sprout initial angle, and sprout final angle (i.e., sprout turning) were recorded. Intriguingly, these analyses revealed that EC sprouts alter their sensitivity to VEGF depending on the matrix density, suggesting a complex interplay between biochemical and biomechanical factors. Steeper VEGF gradients and higher VEGF absolute concentrations are required to induce directional sprouting within stiffer matrices. As angiogenesis progresses, the stalk cells within the multi-cellular sprouts organize into tubular structures with hollow lumens that can transport blood. This process is hypothesized to be abnormal during tumor-initiated angiogenesis, whereby ECs often fail to form tight 5 luminal junctions resulting in leaky blood vessels . To investigate the possible role of local cell density in lumen formation, we quantified the number of cells per sprout
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unit length (Fig. 2I). In stiffer matrices (density = 1.9 mg/ml), the number of cells per 10 microns of sprout length was about 50% higher than in more compliant matrices (density = 1.2 mg/ml) (Fig. 2J). To quantify lumen formation, we defined the Lumen Formation Index as the length of each sprout section along which a lumen is present normalized to the total length of the sprout, as observed using confocal microscopy. In more rigid matrices, z-stack cross-sections of sprouts clearly depict lumen formation along ~45% of the sprout length. In contrast, in more compliant gels, sprout cross-sections rarely depict a clearly defined lumen (Fig. 2K). Interestingly, the higher density of cells per sprout length appears to correlate with the ability to form a stable lumen, with an average of three cells per cross-section present in stiffer gels and two cells per cross-section in more compliant gels. These results suggest that a minimum number of at least three cells may be required to form a lumen with a stable, tube-like geometry. We also quantified the potential saturating effects of VEGF gradients on sprout path-finding. The cell culture chamber of the microfluidic device was divided into three zones transversely, with each zone having an identical gradient steepness of ~50 ng/ml/mm. Zone 1 is furthest from the VEGF source, Zone 2 is in the middle, and Zone 3 is closest to the VEGF source, corresponding to average concentrations of 110, 125, and 140 ng/ml, respectively (Fig. 1A). Within more compliant collagen gels, the final sprout elongation angles were substantially aligned with the VEGF gradient in all three Zones. Similarly, the overall number of sprouts formed in each Zone was statistically identical (Fig. 2L). These results demonstrate that 110 ng/ml is above the saturation threshold for VEGF in these compliant gels. In contrast, within the stiffer collagen gels, the number of sprouts formed and the orientation of each sprout were significantly different across the three Zones. The incidence of sprouting was significantly increased at higher VEGF concentrations (Fig 2M). Similar to the frequency data, final sprout alignment with the VEGF gradient was strongly correlated to the VEGF concentration in the more rigid gels. These data suggest that EC sprouts have a higher VEGF saturation limit in rigid gels compared to more compliant gels.
Figure 2. Morphology and kinetics of EC sprouting within VEGF gradients. Representative phase contrast images of sprout morphology at low (A, B; 0.3 and 0.7 mg/mL), intermediate (C, D; 1.2 and 1.9 mg/mL) and high collagen densities (E; 2.7 mg/mL). F. Number of new sprouts observed at various time points normalized to the total number of observed sprouts. G. Average aspect ratio (sprout length to width). H. Total number of sprouts formed per bead. I. Confocal image of ECs forming multi-cellular sprouts. J. Average number of cells per 10 microns of sprout length. K. Lumen formation index. L, M. Number of sprouts formed within three zones of the device within more compliant (L) and stiffer (M) matrices. Each zone has an identical VEGF gradient steepness and a different average VEGF concentration.
Conclusions: Together, these results demonstrate that matrix stiffness is a mediating factor in guiding the orientation of ECs during sprouting. Corroborating earlier studies, we demonstrate that EC response to VEGF is mediated by matrix rigidity. Our results are strong motivation for the careful consideration of biomechanical factors when designing future biochemical studies of ECs. Our described platform, which can simultaneously and independently modify biomechanical and biochemical stimuli, may be a suitable high-throughput screen for potential pro- and anti-angiogenic regulators with therapeutic applications in wound healing, tumor development, and ischemia treatment.
References: 1. Chen RR. et al. Faseb J. 2007, 21:3896-3903. 2. Sieminski AL. Exp Cell Res. 2004, 297:574-584. 3. Mammoto A. et al. Nature. 2009, 457:1103-1157. 4. Shamloo A. et al. Lab Chip. 2008, 8:1292-1299. 5. Carmeliet P. et al, Nature. 2000, 407:249-257.
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