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1.  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.
PMCID: PMC4096003  PMID: 24580565
angiogenesis; microvessels; microvascular orientation; microvascular remodeling; microvessel guidance; three-dimensional (3D) vascular constructs; matrix mechanics
2.  Dissecting the Role of Human Embryonic Stem Cell–Derived Mesenchymal Cells in Human Umbilical Vein Endothelial Cell Network Stabilization in Three-Dimensional Environments 
Tissue Engineering. Part A  2012;19(1-2):211-223.
The microvasculature is principally composed of two cell types: endothelium and mural support cells. Multiple sources are available for human endothelial cells (ECs) but sources for human microvascular mural cells (MCs) are limited. We derived multipotent mesenchymal progenitor cells from human embryonic stem cells (hES-MC) that can function as an MC and stabilize human EC networks in three-dimensional (3D) collagen-fibronectin culture by paracrine mechanisms. Here, we have investigated the basis for hES-MC-mediated stabilization and identified the pleiotropic growth factor hepatocyte growth factor/scatter factor (HGF/SF) as a putative hES-MC-derived regulator of EC network stabilization in 3D in vitro culture. Pharmacological inhibition of the HGF receptor (Met) (1 μm SU11274) inhibits EC network formation in the presence of hES-MC. hES-MC produce and release HGF while human umbilical vein endothelial cells (HUVEC) do not. When HUVEC are cultured alone the networks collapse, but in the presence of recombinant human HGF or conditioned media from human HGF-transduced cells significantly more networks persist. In addition, HUVEC transduced to constitutively express human HGF also form stable networks by autocrine mechanisms. By enzyme-linked immunosorbent assay, the coculture media were enriched in both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2), but at significantly different levels (Ang1=159±15 pg/mL vs. Ang2=30,867±2685 pg/mL) contributed by hES-MC and HUVEC, respectively. Although the coculture cells formed stabile network architectures, their morphology suggests the assembly of an immature plexus. When HUVEC and hES-MC were implanted subcutaneously in immune compromised Rag1 mice, hES-MC increased their contact with HUVEC along the axis of the vessel. This data suggests that HUVEC and hES-MC form an immature plexus mediated in part by HGF and angiopoietins that is capable of maturation under the correct environmental conditions (e.g., in vivo). Therefore, hES-MC can function as microvascular MCs and may be a useful cell source for testing EC–MC interactions.
doi:10.1089/ten.tea.2011.0408
PMCID: PMC3530951  PMID: 22971005
3.  Microvascular Repair: Post-Angiogenesis Vascular Dynamics 
Microcirculation (New York, N.Y. : 1994)  2012;19(8):10.1111/j.1549-8719.2012.00207.x.
Vascular compromise and the accompanying perfusion deficits cause or complicate a large array of disease conditions and treatment failures. This has prompted the exploration of therapeutic strategies to repair or regenerate vasculatures thereby establishing more competent microcirculatory beds. Growing evidence indicates that an increase in vessel numbers within a tissue does not necessarily promote an increase in tissue perfusion. Effective regeneration of a microcirculation entails the integration of new stable microvessel segments into the network via neovascularization. Beginning with angiogenesis, neovascularization entails an integrated series of vascular activities leading to the formation of a new mature microcirculation and includes vascular guidance and inosculation, vessel maturation, pruning, arterio-venous specification, network patterning, structural adaptation, intussusception, and microvascular stabilization. While the generation of new vessel segments is necessary to expand a network, without the concomitant neovessel remodeling and adaptation processes intrinsic to microvascular network formation, these additional vessel segments give rise to a dysfunctional microcirculation. While many of the mechanisms regulating angiogenesis have been detailed, a thorough understanding of the mechanisms driving post-angiogenesis activities specific to neovascularization has yet to be fully realized, but is necessary in order to develop effective therapeutic strategies for repairing compromised microcirculations as a means to treat disease.
doi:10.1111/j.1549-8719.2012.00207.x
PMCID: PMC3842172  PMID: 22734666
4.  Determinants of Microvascular Network Topologies in Implanted Neovasculatures 
Objectives
During neovascularization, the end result is a new functional microcirculation comprised of a network of mature microvessels with specific topologies. While 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 pre-patterning on the final microvascular network topology using an implant model of implant neovascularization.
Methods and Results
To test this, we used 3-D direct-write bioprinting or physical constraints in a manner permitting post-angiogenesis vascular remodeling and adaptation to pattern angiogenic microvascular precursors (neovessels formed from isolated microvessel segments) in 3-dimensional collagen gels prior to implantation and subsequent network formation. Neovasculatures pre-patterned into parallel arrays formed functional networks following 4 weeks post-implantation, but lost the pre-patterned architecture. However, maintenance of uniaxial physical constraints during post-angiogenesis 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 pre-patterning of precursors but can be influenced by a patterning stimulus involving tissue deformation during post-angiogenesis remodeling and maturation.
doi:10.1161/ATVBAHA.111.238725
PMCID: PMC3256738  PMID: 22053070
microcirculation; regeneration; bioprinting; vascular engineering; neovascularization
5.  Engineering fibrin polymers through engagement of alternative polymerization mechanisms 
Biomaterials  2011;33(2):535-544.
Fibrin is an attractive material for regenerative medicine applications. It not only forms a polymer but also contains cryptic matrikines that are released upon its activation/degradation and enhance the regenerative process. Despite this advantageous biology associated with fibrin, commercially available systems (e.g. TISSEEL) display limited regenerative capacity. This limitation is in part due to formulations that are optimized for tissue sealant applications and result in dense fibrous networks that limit cell infiltration. Recent evidence suggests that polymerization knob ‘B’ engagement of polymerization hole ‘b’ activates an alternative polymerization mechanism in fibrin, which may result in altered single fiber mechanical properties. We hypothesized that augmenting fibrin polymerization through the addition of PEGylated knob peptides with specificity to hole ‘b’ (AHRPYAAC-PEG) would result in distinct fibrin polymer architectures with grossly different physical properties. Polymerization dynamics, polymer architecture, diffusivity, viscoelasticity, and degradation dynamics were analyzed. Results indicate that specific engagement of hole ‘b’ with PEGylated knob ‘B’ conjugates during polymerization significantly enhances the porosity of and subsequent diffusivity through fibrin polymers. Paradoxically, these polymers also display increased viscoelastic properties and decreased susceptibility to degradation. As a result, fibrin polymer strength was significantly augmented without any adverse effects on angiogenesis within the modified polymers.
doi:10.1016/j.biomaterials.2011.09.079
PMCID: PMC3350801  PMID: 22018389
Fibrin; Angiogenesis; Mechanical properties; Biodegradation
6.  Microvascular Mural Cell Functionality of Human Embryonic Stem Cell-Derived Mesenchymal Cells 
Tissue Engineering. Part A  2011;17(11-12):1537-1548.
Microvascular mural or perivascular cells are required for the stabilization and maturation of the remodeling vasculature. However, much less is known about their biology and function compared to large vessel smooth muscle cells. We have developed lines of multipotent mesenchymal cells from human embryonic stem cells (hES-MC); we hypothesize that these can function as perivascular mural cells. Here we show that the derived cells do not form teratomas in SCID mice and independently derived lines show similar patterns of gene expression by microarray analysis. When exposed to platelet-derived growth factor-BB, the platelet-derived growth factor receptor β is activated and hES-MC migrate in response to a gradient. We also show that in a serum-free medium, transforming growth factor β1 (TGFβ1) induces robust expression of multiple contractile proteins (α smooth muscle actin, smooth muscle myosin heavy chain, smooth muscle 22α, and calponin). TGFβ1 signaling is mediated through the TGFβR1/Alk5 pathway as demonstrated by inhibition of α smooth muscle actin expression by treatment of the Alk5-specific inhibitor SB525334 and stable retroviral expression of the Alk5 dominant negative (K232R). Coculture of human umbilical vein endothelial cell (HUVEC) with hES-MC maintains network integrity compared to HUVEC alone in three-dimensional collagen I-fibronectin by paracrine signaling. Using high-resolution laser confocal microscopy, we show that hES-MC also make direct contact with HUVEC. This demonstrates that hESC-derived mesenchymal cells possess the molecular machinery expected in a perivascular progenitor cells and can play a functional role in stabilizing EC networks in in vitro three-dimensional culture.
doi:10.1089/ten.tea.2010.0397
PMCID: PMC3098949  PMID: 21284534
7.  Angiogenic potential of microvessel fragments is independent of the tissue of origin and can be influenced by the cellular composition of the implants 
We have demonstrated that microvessel fragments (MFs) isolated from adipose retain angiogenic potential in vitro and form a mature, perfused network when implanted. However, adipose-derived microvessels are rich in pro-vascularizing cells that could uniquely drive neovascularization in adipose-derived MFs implants.
Objective
Investigate the ability of microvessel fragments from a different vascular bed to recapitulate adipose-derived microvessel angiogenesis and network formation and analyze adipose-derived vessel plasticity by assessing whether vessel function could be modulated by astrocyte-like cells.
Methods
MFs were isolated by limited collagenase digestion from rodent brain or adipose and assembled into 3D collagen gels in the presence or absence of GRPs. The resulting neovasculatures that formed following implantation were assessed by measuring 3-D vascularity and vessel permeability to small and large molecular tracers.
Results
Similar to adipose-derived MFs, brain-derived MFs can sprout and form a perfused neovascular network when implanted. Furthermore, when co-implanted in the constructs, GRPs caused adipose-derived vessels to express the brain endothelial marker glucose transporter-1 and to significantly reduce microvessel permeability.
Conclusion
Neovascularization involving isolated microvessel elements is independent of the tissue origin and degree of vessel specialization. In addition, adipose-derived vessels have the ability to respond to environmental signals and change vessel characteristics.
doi:10.1111/j.1549-8719.2010.00052.x
PMCID: PMC3057771  PMID: 21040121
Angiogenesis; vessel permeability; glial restricted precursors; astrocytes; angiogenesis assay
8.  Angiogenesis in a Microvascular Construct for Transplantation Depends on the Method of Chamber Circulation 
Tissue Engineering. Part A  2009;16(3):795-805.
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-1α. By altering circulation conditions in the DIP chamber, we can control, possibly by modulating hypoxic stress, prevascularizing activity in vitro.
doi:10.1089/ten.tea.2009.0370
PMCID: PMC2862615  PMID: 19778185
9.  Effect of Mechanical Boundary Conditions on Orientation of Angiogenic Microvessels 
Cardiovascular research  2008;78(2):324-332.
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
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).
Results
Neovessels sprouted and grew by the 3rd 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 to the free-floating shape control, no stretch or cyclic stretch groups. In all mechanically loaded cultures, neovessel diameter and length distributions were heterogeneous, while 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 due to collagen fibril alignment induced by the growing neovessels themselves.
doi:10.1093/cvr/cvn055
PMCID: PMC2840993  PMID: 18310100
boundary conditions; angiogenesis; strain; orientation; morphometry; image analysis
10.  Interaction of Angiogenic Microvessels with the Extracellular Matrix 
The extracellular matrix (ECM) plays a critical role in angiogenesis by providing biochemical and positional cues as well as by mechanically influencing microvessel cell behavior. Considerable information is known concerning the biochemical cues relevant to angiogenesis, but less is known about the mechanical dynamics during active angiogenesis. The objective of this study was to characterize changes in the material properties of a simple angiogenic tissue before and during angiogenesis. During sprouting, there was an overall decrease in tissue stiffness followed by an increase during neovessel elongation. The fall in matrix stiffness coincided with peak MMP mRNA expression and elevated proteolytic activity. An elevated expression of genes for ECM componenets and cell-ECM interaction molecules and a subsequent drop in proteolytic activity (although enzyme levels remained elevated) coincided with the subsequent stiffening.. The results of this study show that the mechanical properties of a scaffold tissue may be actively modified during angiogenesis by the growing microvasculature.
doi:10.1152/ajpheart.00772.2007
PMCID: PMC2840990  PMID: 17933969
angiogenesis; biomechanics; matrix metalloprotease

Results 1-10 (10)