Contractile forces in platelet aggregates under microfluidic shear gradients reflect platelet inhibition and bleeding risk.

Platelets contract forcefully after their activation, contributing to the strength and stability of platelet aggregates and fibrin clots during blood coagulation. Viscoelastic approaches can be used to assess platelet-induced clot strengthening, but they require thrombin and fibrin generation and are unable to measure platelet forces directly. Here, we report a rapid, microfluidic approach for measuring the contractile force of platelet aggregates for the detection of platelet dysfunction. We find that platelet forces are significantly reduced when blood samples are treated with inhibitors of myosin, GPIb-IX-V, integrin αIIbß3, P2Y12, or thromboxane generation. Clinically, we find that platelet forces are measurably lower in cardiology patients taking aspirin. We also find that measuring platelet forces can identify Emergency Department trauma patients who subsequently require blood transfusions. Together, these findings indicate that microfluidic quantification of platelet forces may be a rapid and useful approach for monitoring both antiplatelet therapy and traumatic bleeding risk.

Control of vascular tube morphogenesis and maturation in 3D extracellular matrices by endothelial cells and pericytes.

An important advance using in vitro EC tube morphogenesis and maturation models has been the development of systems using serum-free defined media. Using this approach, the growth factors and cytokines which are actually necessary for these events can be determined. The first model developed by our laboratory was such a system where we showed that phorbol ester was needed in order to promote survival and tube morphogenesis in 3D collagen matrices. Recently, we have developed a new system in which the hematopoietic stem cell cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and stromal derived factor-1α (SDF-1α) were added in conjunction with FGF-2 to promote human EC tube morphogenesis in 3D collagen matrices under serum-free defined conditions. This new model using SCF, IL-3, SDF-1α, and FGF-2 also works well following the addition of pericytes where EC tube formation occurs, pericytes are recruited to the tubes, and vascular basement membrane matrix assembly occurs following EC-pericyte interactions. In this chapter, we describe several in vitro assay models that we routinely utilize to investigate the molecular requirements that are critical to EC tube formation and maturation events in 3D extracellular matrix environments.

Aligned human microvessels formed in 3D fibrin gel by constraint of gel contraction.

This study aimed to form microvessels in fibrin gels, which is of interest both for studying the fundamental cell-matrix interactions as well as for tissue engineering purposes, and to align the microvessels, which would provide natural inlet and outlet sides for perfusion. The data reported here demonstrate the formation of highly interconnected microvessels in fibrin gel under defined medium conditions and the ability to align them using two methods, both of which involved anchoring the gel at both ends to constrain the cell-induced compaction. The first method used only defined medium and resulted in moderate alignment. The second method used defined and serum-containing media sequentially to achieve high levels of microvessel alignment.

Hematopoietic stem cell cytokines and fibroblast growth factor-2 stimulate human endothelial cell-pericyte tube co-assembly in 3D fibrin matrices under serum-free defined conditions.

We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC) tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF)-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay) to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5ß1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices) that regulates angiogenic events in postnatal life.

Dynamic regulation of the cerebral cavernous malformation pathway controls vascular stability and growth.

Cardiovascular growth must balance stabilizing signals required to maintain endothelial connections and network integrity with destabilizing signals that enable individual endothelial cells to migrate and proliferate. The cerebral cavernous malformation (CCM) signaling pathway utilizes the adaptor protein CCM2 to strengthen endothelial cell junctions and stabilize vessels. Here we identify a CCM2 paralog, CCM2L, that is expressed selectively in endothelial cells during periods of active cardiovascular growth. CCM2L competitively blocks CCM2-mediated stabilizing signals biochemically, in cultured endothelial cells, and in developing mice. Loss of CCM2L reduces endocardial growth factor expression and impairs tumor growth and wound healing. Our studies identify CCM2L as a molecular mechanism by which endothelial cells coordinately regulate vessel stability and growth during cardiovascular development, as well as postnatal vessel growth.