James B. Hoying

Fibroblast growth factor 2 control of vascular tone

Vascular tone control is essential in blood pressure regulation, shock, ischemia-reperfusion, inflammation, vessel injury/repair, wound healing, temperature regulation, digestion, exercise physiology, and metabolism. Here we show that a well-known growth factor, FCF2, long thought to be involved in many developmental and homeostatic processes, including growth of the tissue layers of vessel walls, functions in vascular tone control. Fgf2 knockout mice are morphologically normal and display decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. Following intra-arterial mechanical injury, FGF2-deficient vessels undergo a normal hyperplastic response. These results force us to reconsider the function of FGF2 in vascular development and homeostasis in terms of vascular tone control.

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.

Angiogenic potential of microvessel fragments established in three-dimensional collagen gels.

SummaryDuring angiogenesis, the microvasculature displays both vessel remodeling and expansion under the control of both cellular and extracellular influences. We have evaluated the role of angiogenic and angiostatic molecules on angiogenesis in anin vitro model that more appropriately duplicates the cellular and extracellular components of this process. Freshly isolated microvessel fragments from rat adipose tissue (RFMF) were cultured within three-dimensional collagen I gels. These fragments were characterized at the time of isolation and were composed of vessel segments observed in the microvasculature of fatin situ (i.e., arterioles, venules, and capillaries). Fragments also exhibited characteristic ablumenally associated cells including smooth muscle cells and pericytes. Finally, fragments were encased in an extracellular matrix composed of collagen type IV and collagen type I/III. The elongation of microvascular elements was subsequently evaluated using morphologic and immunocytochemical techniques. The proliferation, migration, and elongation of cellular elements in microvessel fragments from rat adipose tissue was dependent on initial fragment density, matrix density, and required serum. Inclusion of endothelial cell growth factors to microvessel fragments from rat adipose tissue 3-D cultures resulted in the accelerated elongation of tube structures and the expression of von Willebrand factor in cells constituting these tubes. Molecules with reported angiostatic capacity (e.g., transforming growth factor and hydrocortisone) inhibited vessel tube elongation. In vitro methods have been developed to evaluate numerous mechanisms associated with angiogenesis, including endothelial cell proliferation, migration, and phenotypic modulation. Microvascular endothelial cell fragments described in this study represent anin vitro population of cells that accurately duplicate thein vivo microcirculatory elements of fat. The proliferation of cells and elongation of microvascular elements subsequently observed in three-dimensional cultures provides anin vitro model of angiogenesis. Microvascular formation in this system results from pre-existing microvessel fragments unlike tube formation observed when cultured endothelial cells are placed in three-dimensional gels. This form of tube formation from cultured endothelium is more characteristic of vasculogenesis. Thus, the formation of microvascular elements from microvessel fragments provides the opportunity to examine the mechanisms regulating angiogenesis in anin vitro system amenable to precise experimental manipulation.

Defective endothelium-dependent relaxation of vascular smooth muscle and endothelial cell Ca2+ signaling in mice lacking sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3.

Sarco(endo)plasmic reticulum Ca2+ ATPase isoform 3 (SERCA3) is one of two Ca2+ pumps serving intracellular Ca2+ signaling pools in non-muscle tissues; however, unlike the ubiquitous SERCA2b, it exhibits a restricted cell-type distribution. Gene targeting was used to generate a mouse with a null mutation in the SERCA3 gene. Homozygous mutant mice were viable, fertile, and did not exhibit an overt disease phenotype. Because SERCA3 is expressed in arterial endothelial cells, aortic ring preparations were analyzed to determine whether it is involved in the regulation of vascular tone. Contraction-isometric force relations in response to phenylephrine or KCl, as well as relaxation produced by exposure to a nitric oxide donor, were similar in wild-type and null mutant aortas. Acetylcholine-induced endothelium-dependent relaxation of aortas after precontraction with phenylephrine was significantly reduced in homozygous mutants (61.3 ± 5.6% in wild type, 35.4 ± 7.3% in mutants). Ca2+ imaging of cultured aortic endothelial cells demonstrated that the acetylcholine-induced intracellular Ca2+ signal is sharply diminished in SERCA3-deficient cells and also indicated that replenishment of the acetylcholine-responsive Ca2+ stores is severely impaired. These results indicate that SERCA3 plays a critical role in endothelial cell Ca2+signaling events involved in nitric oxide-mediated relaxation of vascular smooth muscle.

Rapid Perfusion and Network Remodeling in a Microvascular Construct After Implantation

Objective—We have previously demonstrated the ability to construct 3-dimensional microvascular beds in vitro via angiogenesis from isolated, intact, microvessel fragments that retain endothelial cells and perivascular cells. Our objective was to develop and characterize an experimental model of tissue vascularization, based on the implantation of this microvascular construct, which recapitulated angiogenesis, vessel differentiation, and network maturation. Methods and Results—On implantation in a severe combined-immunodeficient mouse model, vessels in the microvascular constructs rapidly inosculated with the recipient host circulation. Ink perfusion of implants via the left ventricle of the host demonstrated that vessel inosculation begins within the first day after implantation. Evaluation of explanted constructs over the course of 28 days revealed the presence of a mature functional microvascular bed. Using a probe specific for the original microvessel source, 91.7%±11% and 88.6%±19% of the vessels by day 5 and day 28 after implantation, respectively, were derived from the original microvessel isolate. Similar results were obtained when human-derived microvessels were used to build the microvascular construct. Conclusions—With this model, we reproduce the important aspects of vascularization, angiogenesis, inosculation, and network remodeling. Furthermore, we demonstrate that the model accommodates human-derived vessel fragments, enabling the construction of human–mouse vascular chimeras.

Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier

We describe a method for generating primary cultures of human brain microvascular endothelial cells (HBMVECs). HBMVECs are derived from microvessels isolated from temporal tissue removed during operative treatment of epilepsy. The tissue is mechanically fragmented and size filtered using polyester meshes. The resulting microvessel fragments are placed onto type I collagen-coated flasks to allow HBMVECs to migrate and proliferate. The overall process takes less than 3 h and does not require specialized equipment or enzymatic processes. HBMVECs are typically cultured for approximately 1 month until confluent. Cultures are highly pure (∼97% endothelial cells; ∼3% pericytes), are reproducible, and show characteristic brain endothelial markers (von Willebrand factor, glucose transporter-1) and robust expression of tight and adherens junction proteins as well as caveolin-1 and efflux protein P-glycoprotein. Monolayers of HBMVECs show characteristically high transendothelial electric resistance and have proven useful in multiple functional studies for in vitro modeling of the human blood-brain barrier.

Effect of mechanical boundary conditions on orientation of angiogenic microvessels

AIM Mechanical forces are important regulators of cell and tissue phenotype. We hypothesized that mechanical loading and boundary conditions would influence neovessel activity during angiogenesis. METHODS AND RESULTS Using an in vitro model of angiogenesis sprouting and a mechanical loading system, we evaluated the effects of boundary conditions and applied loading. The model consisted of rat microvessel fragments cultured in a 3D collagen gel, previously shown to recapitulate angiogenic sprouting observed in vivo. We examined changes in neovascular growth in response to four different mechanical conditions. Neovessel density, diameter, length and orientation were measured from volumetric confocal images of cultures exposed to no external load (free-floating shape control), intrinsic loads (fixed ends, no stretch), static external load (static stretch), or cyclic external load (cyclic stretch). Neovessels sprouted and grew by the third day of culture and continued to do so during the next 3 days of loading. The numbers of neovessels and branch points were significantly increased in the static stretch group when compared with the free-floating shape control group. In all mechanically loaded cultures, neovessel diameter and length distributions were heterogeneous, whereas they were homogeneous in shape control cultures. Neovessels were significantly more oriented along the direction of mechanical loading than those in the shape controls. Interestingly, collagen fibrils were organized parallel and adjacent to growing neovessels. CONCLUSION Externally applied boundary conditions regulate neovessel sprouting and elongation during angiogenesis, affecting both neovessel growth characteristics and network morphometry. Furthermore, neovessels align parallel to the direction of stress/strain or internally generated traction, and this may be because of collagen fibril alignment induced by the growing neovessels themselves.

Design and Application of a Test System for Viscoelastic Characterization of Collagen Gels

Characterization and control of the mechanical properties of the extracellular matrix are critical to the interpretation of results of in vitro studies of cultured tissues and cells and for the design of functional engineered constructs. In this work a viscoelastic tensile test system and custom culture chambers were developed and characterized. The system allowed quantification of strain as well as the stresses developed during cyclic viscoelastic material testing. Finite element analysis of the culture chambers indicated that the tensile strains near the actuated ends of the gel were greater than the strains experienced by material in the center of the culture chambers. However, the strain was uniformly distributed over the central substance of the gel, validating the assumption that a homogeneous strain state existed in the central region of the chamber. Viscoelastic testing was performed on collagen gels that were created with three different collagen concentrations. Results demonstrated that there was a significant increase in the dynamic stiffness of the gels with increasing equilibrium strain, collagen concentration, and frequency of applied strain. With increasing strain rate, the phase angle, representing the energy dissipated, dropped initially and then increased at higher rates. Mechanical testing of gels at different time intervals up to 7 days after polymerization demonstrated that the material properties remained stable when appropriate environmental conditions were maintained. The ability to characterize the viscoelastic properties of gels after different periods of culture will allow the quantification of alterations in gel material properties due to changes in cell cytoskeletal organization, cell-matrix interactions, and cellular activity on the matrix. Further, the test device provides a means to apply controlled mechanical loading to growing gel cultures. Finally, the results of this study will provide guidance to the design of further experiments on this substrate.

Imaging of the Ovary

Epithelial ovarian cancer has the highest mortality rate among the gynecologic cancers and spreads beyond the ovary in 90% of the women diagnosed with ovarian cancer. Detection before the disease has spread beyond the ovary would significantly improve the survival from ovarian cancer, which is currently only 30% over 5 years, despite extensive efforts to improve the survival. This study describes initial investigation of the use of optical technologies to improve the outcome for this disease by detecting cancers at an earlier and more treatable stage. Women undergoing oophorectomy were recruited for this study. Ovaries were harvested for fluorescence spectroscopy, confocal microscopy, and optical coherence tomography. Fluorescence spectroscopy showed large diagnostic differences between normal and abnormal tissue at 270 and 340 nm excitation. Optical coherence tomography was able to image up to 2mm deep into the ovary with particular patterns of backscattered intensity observed in normal versus abnormal tissue. Fluorescence confocal microscopy was able to visualize sub-cellular structures of the surface epithelium and underlying cell layers. Optical imaging and/or spectroscopy has the potential to improve the diagnostic capability in the ovary, but extended systematic investigations are needed to identify the unique signatures of disease. The combination of optical technologies supported by modern molecular biology may lead to an instrument that can accurately detect early carcinogenesis.

Flow-Dependent Remodeling in the Carotid Artery of Fibroblast Growth Factor-2 Knockout Mice

Objective—Fibroblast growth factor-2 (FGF2) has been implicated as a mediator in the structural remodeling of arteries. Chronic changes in blood flow are known to cause reorganization of the vessel wall, resulting in permanent changes in artery size (flow-dependent remodeling). Using FGF2 knockout (Fgf2−/−) mice, we tested the hypothesis that FGF2 is required during flow-dependent remodeling of the carotid arteries. Methods and Results—All branches originating from the left common carotid artery (LCCA), except for the left thyroid artery, were ligated to reduce flow in the LCCA and increase flow in the contralateral right common carotid artery (RCCA). Age- and sex-matched control animals did not undergo ligation of the LCCA branches. Morphometric analysis showed that by day 7, vessel diameter was significantly greater in the high-flow RCCA of FGF2 wild-type (Fgf2+/+) and Fgf2−/− mice versus the respective control RCCA, demonstrating outward remodeling. In contrast, vessel diameter was decreased by day 7 in the low-flow LCCA of both genotypes compared with the control LCCA, showing inward remodeling. No differences were observed between Fgf2+/+ and Fgf2−/− mice in either high-flow or low-flow remodeling. Conclusions—Given these results, we demonstrate that FGF2 is not essential for flow-dependent remodeling of the carotid arteries.