The role of cyclic AMP in normalizing the function of engineered human blood microvessels in microfluidic collagen gels

Abstract

Nearly all engineered tissues must eventually be vascularized to survive. To this end, we and others have recently developed methods to synthesize extracellular matrix-based scaffolds that contain open microfluidic networks. These scaffolds serve as templates for the formation of endothelial tubes that can be perfused; whether such microvascular structures are stable and/or functional is largely unknown. Here, we show that compounds that elevate intracellular concentrations of the second messenger cyclic AMP (cAMP) strongly normalize the phenotype of engineered human microvessels in microfluidic type I collagen gels. Cyclic AMP-elevating agents promoted vascular stability and barrier function, and reduced cellular turnover. Under conditions that induced the highest levels of cAMP, the physiology of engineered microvessels in vitro quantitatively mirrored that of native vessels in vivo. Computational analysis indicated that cAMP stabilized vessels partly via its enhancement of barrier function.

Physical and Chemical Signals That Promote Vascularization of Capillary-Scale Channels

“…6, 38 We used genipin to stiffen type I collagen scaffolds from an elastic modulus of ~260 Pa to ~1330 Pa. This degree of stiffening has been shown previously to improve vascular stability of large endothelial tubes.…”

“…These two cAMP concentrations were chosen since they resulted in marked differences in phenotype and vascular stability of large (~120-μm-diameter) engineered microvessels. 26, 38 …”

“…Because endothelial cells migrate as a sheet against the direction of fluid flow (i.e., upstream) 8, 22 , we hypothesized that seeded cells that plugged a narrow channel would migrate to barren areas if flow was directed from barren to plugged regions. Because a cell-permeant analog of cyclic AMP (dibutyryl cAMP) improves the stability of large (~120-μm-diameter) engineered microvessels 26, 38 , we hypothesized that elevated levels of intracellular cAMP would likewise improve the stability of newly formed endothelium in narrow (≤30-μm-diameter) channels. Moreover, substratum stiffness is a strong controller of endothelial cell function 4 ; in particular, stiff substrata promote vascular stability in ~120-μm-diameter channels 6 , and we expected the same to hold at the capillary scale.…”

In vitro microvessels for the study of angiogenesis and thrombosis

“…Important progress has been made toward this goal: Biologically derived or synthetic materials have been used to generate macrovessel tubes (6) and endothelialized microtubes (7); cellular self-assembly has been used to generate random microvasculature (8); microfabrication has been used to define complex geometries in hydrogels at the micro-scale (9); and distributions of cells and biochemical factors within 3D scaffolds (10). Of particular note, the group of Tien has pioneered the use of collagen to template the growth of vascular endothelium (7,11) and demonstrated appropriate permeability (7), response to cyclic AMP (12), and differential properties as a function of the luminal shear stress and composition of the medium (13). Nonetheless, prior methodologies have been unable to produce endothelialized networks that can undergo substantial remodeling via angiogenesis; elucidate the roles of perivascular cells in modulating vascular morphology and permeability; control cellular, chemical, and physical stimuli at microscale levels to manipulate the interactions between different cell types and their matrices; and produce vessels with normal antithrombotic function when quiescent and prothrombotic behavior when exposed to inflammatory stimuli.…”

Collagen-based brain microvasculature modelin vitrousing three-dimensional printed template

“…47) as similarly reported. 33 We operated our microfluidic culture with gravity-driven perfusion leading to relatively high flow rate and thus high P eclet number (Pe) to avoid depletion of nutrients and oxygen to brain endothelial cells in downstream of 8-10 mm-long microchannels. 12 Based on our calculation, Pe was $10 3 , much larger than L/d $ 10, where L and d are length and diameter of a collagen microchannel, respectively.…”