The NHLBI-sponsored Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR): a new paradigm for rigorous, accurate, and reproducible evaluation of putative infarct-sparing interventions in mice, rabbits, and pigs.

RATIONALE: Despite 4 decades of intense effort and substantial financial investment, the cardioprotection field has failed to deliver a single drug that effectively reduces myocardial infarct size in patients. A major reason is insufficient rigor and reproducibility in preclinical studies. OBJECTIVE: To develop a multicenter, randomized, controlled, clinical trial-like infrastructure to conduct rigorous and reproducible preclinical evaluation of cardioprotective therapies. METHODS AND RESULTS: With support from the National Heart, Lung, and Blood Institute, we established the Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR), based on the principles of randomization, investigator blinding, a priori sample size determination and exclusion criteria, appropriate statistical analyses, and assessment of reproducibility. To validate CAESAR, we tested the ability of ischemic preconditioning to reduce infarct size in 3 species (at 2 sites/species): mice (n=22-25 per group), rabbits (n=11-12 per group), and pigs (n=13 per group). During this validation phase, (1) we established protocols that gave similar results between centers and confirmed that ischemic preconditioning significantly reduced infarct size in all species and (2) we successfully established a multicenter structure to support CAESAR's operations, including 2 surgical centers for each species, a Pathology Core (to assess infarct size), a Biomarker Core (to measure plasma cardiac troponin levels), and a Data Coordinating Center-all with the oversight of an external Protocol Review and Monitoring Committee. CONCLUSIONS: CAESAR is operational, generates reproducible results, can detect cardioprotection, and provides a mechanism for assessing potential infarct-sparing therapies with a level of rigor analogous to multicenter, randomized, controlled clinical trials. This is a revolutionary new approach to cardioprotection. Importantly, we provide state-of-the-art, detailed protocols ("CAESAR protocols") for measuring infarct size in mice, rabbits, and pigs in a manner that is rigorous, accurate, and reproducible.

Therapeutic potential of sustained-release sodium nitrite for critical limb ischemia in the setting of metabolic syndrome.

Nitrite is a storage reservoir of nitric oxide that is readily reduced to nitric oxide under pathological conditions. Previous studies have demonstrated that nitrite levels are significantly reduced in cardiovascular disease states, including peripheral vascular disease. We investigated the cytoprotective and proangiogenic actions of a novel, sustained-release formulation of nitrite (SR-nitrite) in a clinically relevant in vivo swine model of critical limb ischemia (CLI) involving central obesity and metabolic syndrome. CLI was induced in obese Ossabaw swine (n = 18) by unilateral external iliac artery deployment of a full cross-sectional vessel occlusion device positioned within an endovascular expanded polytetrafluoroethylene-lined nitinol stent-graft. At post-CLI day 14, pigs were randomized to placebo (n = 9) or SR-nitrite (80 mg, n = 9) twice daily by mouth for 21 days. SR-nitrite therapy increased nitrite, nitrate, and S-nitrosothiol in plasma and ischemic skeletal muscle. Oxidative stress was reduced in ischemic limb tissue of SR-nitrite- compared with placebo-treated pigs. Ischemic limb tissue levels of proangiogenic growth factors were increased following SR-nitrite therapy compared with placebo. Despite the increases in cytoprotective and angiogenic signals with SR-nitrite therapy, new arterial vessel formation and enhancement of blood flow to the ischemic limb were not different from placebo. Our data clearly demonstrate cytoprotective and proangiogenic signaling in ischemic tissues following SR-nitrite therapy in a very severe model of CLI. Further studies evaluating longer-duration nitrite therapy and/or additional nitrite dosing strategies are warranted to more fully evaluate the therapeutic potential of nitrite therapy in peripheral vascular disease.

Manipulating the microvasculature and its microenvironment.

The microvasculature is a dynamic cellular system necessary for tissue health and function. Therapeutic strategies that target the microvasculature are expanding and evolving, including those promoting angiogenesis and microvascular expansion. When considering how to manipulate angiogenesis, either as part of a tissue construction approach or a therapy to improve tissue blood flow, it is important to know the microenvironmental factors that regulate and direct neovessel sprouting and growth. Much is known concerning both diffusible and matrix-bound angiogenic factors, which stimulate and guide angiogenic activity. How the other aspects of the extravascular microenvironment, including tissue biomechanics and structure, influence new vessel formation is less well known. Recent research, however, is providing new insights into these mechanisms and demonstrating that the extent and character of angiogenesis (and the resulting new microcirculation) is significantly affected. These observations and the resulting implications with respect to tissue construction and microvascular therapy are addressed.

Determinants of microvascular network topologies in implanted neovasculatures.

OBJECTIVE: During neovascularization, the end result is a new functional microcirculation composed of a network of mature microvessels with specific topologies. Although much is known concerning the mechanisms underlying the initiation of angiogenesis, it remains unclear how the final architecture of microcirculatory beds is regulated. To begin to address this, we determined the impact of angiogenic neovessel prepatterning on the final microvascular network topology using a model of implant neovascularization. METHODS AND RESULTS: We used 3D direct-write bioprinting or physical constraints in a manner permitting postangiogenesis vascular remodeling and adaptation to pattern angiogenic microvascular precursors (neovessels formed from isolated microvessel segments) in 3D collagen gels before implantation and subsequent network formation. Neovasculatures prepatterned into parallel arrays formed functional networks after 4 weeks postimplantation but lost the prepatterned architecture. However, maintenance of uniaxial physical constraints during postangiogenesis remodeling of the implanted neovasculatures produced networks with aligned microvessels, as well as an altered proportional distribution of arterioles, capillaries, and venules. CONCLUSIONS: Here we show that network topology resulting from implanted microvessel precursors is independent from prepatterning of precursors but can be influenced by a patterning stimulus involving tissue deformation during postangiogenesis remodeling and maturation.

Directed three-dimensional growth of microvascular cells and isolated microvessel fragments.

Tissue engineering has promise as a means for repairing diseased and damaged tissues. A significant challenge in tissue construction relates to the constraints placed on tissue geometries resulting from diffusion limitations. An ability to incorporate a premade vasculature would overcome these difficulties and promote construct viability once implanted. Most in vitro microvascular fabrication strategies rely on surface-associated cell growth, manipulated cell monolayers, or random arrangement of cells within matrix materials. In contrast, we successfully suspended microvascular cells and isolated microvessel fragments within collagen and then microfluidically drove the mixtures into microfabricated network topologies. Developing within the 3D collagen matrix, patterned cells progressed into cord-like morphologies. These geometries were directed by the surrounding elastomer mold. With similar techniques, suspended fragments formed endothelial sprouts. By avoiding the addition of exogenous growth factors, we allowed constituent cells and fragments to autonomously develop within the constructs, providing a more physiologically relevant system for in vitro microvascular development. In addition, we present the first examples of directed endothelial cell sprouting from parent microvessel fragments. We believe this system may serve as a foundation for future in vivo fabrication of microvascular networks for tissue engineering applications.

Intimal hyperplasia following implantation of helical-centreline and straight-centreline stents in common carotid arteries in healthy pigs: influence of intraluminal flow.

Intimal hyperplasia (IH) is a leading cause of obstruction of vascular interventions, including arterial stents, bypass grafts and arteriovenous grafts and fistulae. Proposals to account for arterial stent-associated IH include wall damage, low wall shear stress (WSS), disturbed flow and, although not widely recognized, wall hypoxia. The common non-planarity of arterial geometry and flow, led us to develop a bare-metal, nitinol, self-expanding stent with three-dimensional helical-centreline geometry. This was deployed in one common carotid artery of healthy pigs, with a straight-centreline, but otherwise identical (conventional) stent deployed contralaterally. Both stent types deformed the arteries, but the helical-centreline device additionally deformed them helically and caused swirling of intraluminal flow. At sacrifice, one month post stent deployment, histology revealed significantly less IH in the helical-centreline than straight-centreline stented vessels. Medial cross-sectional area was not significantly different in helical-centreline than straight-centreline stented vessels. By contrast, luminal cross-sectional area was significantly larger in helical-centreline than straight-centreline stented vessels. Mechanisms considered to account for those results include enhanced intraluminal WSS and enhanced intraluminal blood-vessel wall mass transport, including of oxygen, in the helical-centreline stented vessels. Consistent with the latter proposal, adventitial microvessel density was lower in the helical-centreline stented than straight-centreline stented vessels.

Direct-write bioprinting three-dimensional biohybrid systems for future regenerative therapies.

Regenerative medicine seeks to repair or replace dysfunctional tissues with engineered biological or biohybrid systems. Current clinical regenerative models utilize simple uniform tissue constructs formed with cells cultured onto biocompatible scaffolds. Future regenerative therapies will require the fabrication of complex three-dimensional constructs containing multiple cell types and extracellular matrices. We believe bioprinting technologies will provide a key role in the design and construction of future engineered tissues for cell-based and regenerative therapies. This review describes the current state-of-the-art bioprinting technologies, focusing on direct-write bioprinting. We describe a number of process and device considerations for successful bioprinting of composite biohybrid constructs. In addition, we have provided baseline direct-write printing parameters for a hydrogel system (Pluronic F127) often used in cardiovascular applications. Direct-write dispensed lines (gels with viscosities ranging from 30 mPa s to greater than 600 × 106 mPa s) were measured following mechanical and pneumatic printing via three commercially available needle sizes (20 ga, 25 ga, and 30 ga). Example patterns containing microvascular cells and isolated microvessel fragments were also bioprinted into composite 3D structures. Cells and vessel fragments remained viable and maintained in vitro behavior after incorporation into biohybrid structures. Direct-write bioprinting of biologicals provides a unique method to design and fabricate complex, multicomponent 3D structures for experimental use. We hope our design insights and baseline parameter descriptions of direct-write bioprinting will provide a useful foundation for colleagues to incorporate this 3D fabrication method into future regenerative therapies.

Vessel arterial-venous plasticity in adult neovascularization.

OBJECTIVE: Proper arterial and venous specification is a hallmark of functional vascular networks. While arterial-venous identity is genetically pre-determined during embryo development, it is unknown whether an analogous pre-specification occurs in adult neovascularization. Our goal is to determine whether vessel arterial-venous specification in adult neovascularization is pre-determined by the identity of the originating vessels. METHODS AND RESULTS: We assessed identity specification during neovascularization by implanting isolated microvessels of arterial identity from both mice and rats and assessing the identity outcomes of the resulting, newly formed vasculature. These microvessels of arterial identity spontaneously formed a stereotypical, perfused microcirculation comprised of the full complement of microvessel types intrinsic to a mature microvasculature. Changes in microvessel identity occurred during sprouting angiogenesis, with neovessels displaying an ambiguous arterial-venous phenotype associated with reduced EphrinB2 phosphorylation. CONCLUSIONS: Our findings indicate that microvessel arterial-venous identity in adult neovascularization is not necessarily pre-determined and that adult microvessels display a considerable level of phenotypic plasticity during neovascularization. In addition, we show that vessels of arterial identity also hold the potential to undergo sprouting angiogenesis.

Angiogenesis in a microvascular construct for transplantation depends on the method of chamber circulation.

Effective tissue prevascularization depends on new vessel growth and subsequent progression of neovessels into a stable microcirculation. Isolated microvessel fragments in a collagen-based microvascular construct (MVC) spontaneously undergo angiogenesis in static conditions in vitro but form a new microcirculation only when implanted in vivo. We have designed a bioreactor, the dynamic in vitro perfusion (DIP) chamber, to culture MVCs in vitro with perfusion. By altering bioreactor circulation, microvessel fragments in the DIP chamber either maintained stable, nonsprouting, patent vessel morphologies or sprouted endothelial neovessels that extended out into the surrounding collagen matrix (i.e., angiogenesis), yielding networks of neovessels within the MVC. Neovessels formed in regions of the construct predicted by simulation models to have the steepest gradients in oxygen levels and expressed hypoxia inducible factor-1alpha. By altering circulation conditions in the DIP chamber, we can control, possibly by modulating hypoxic stress, prevascularizing activity in vitro.