Mechanical forces are important regulators of cell and tissue phenotype. We hypothesized that mechanical loading and boundary conditions would influence neovessel activity during angiogenesis.
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 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.
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.
boundary conditions; angiogenesis; strain; orientation; morphometry; image analysis
Angiogenesis is regulated by the local microenvironment, including the mechanical interactions between neovessel sprouts and the extracellular matrix (ECM). However, the mechanisms controlling the relationship of mechanical and biophysical properties of the ECM to neovessel growth during sprouting angiogenesis are just beginning to be understood. In this research, we characterized the relationship between matrix density and microvascular topology in an in vitro 3D organ culture model of sprouting angiogenesis. We used these results to design and calibrate a computational growth model to demonstrate how changes in individual neovessel behavior produce the changes in vascular topology that were observed experimentally. Vascularized gels with higher collagen densities produced neovasculatures with shorter vessel lengths, less branch points, and reduced network interconnectivity. The computational model was able to predict these experimental results by scaling the rates of neovessel growth and branching according to local matrix density. As a final demonstration of utility of the modeling framework, we used our growth model to predict several scenarios of practical interest that could not be investigated experimentally using the organ culture model. Increasing the density of the ECM significantly reduced angiogenesis and network formation within a 3D organ culture model of angiogenesis. Increasing the density of the matrix increases the stiffness of the ECM, changing how neovessels are able to deform and remodel their surroundings. The computational framework outlined in this study was capable of predicting this observed experimental behavior by adjusting neovessel growth rate and branching probability according to local ECM density, demonstrating that altering the stiffness of the ECM via increasing matrix density affects neovessel behavior, thereby regulated vascular topology during angiogenesis.
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.
During the typical healing response to an implanted biomaterial, vascular-rich granulation tissue forms around the implant and later resolves into a relatively avascular, fibrous capsule. We have previously shown that a microvascular construct (MVC) consisting of isolated microvessel fragments suspended in a collagen I gel forms a persistent microcirculation in lieu of avascular scar when implanted. The current study evaluated the potential for microvascular constructs to maintain a vascularized tissue environment around an implanted biomaterial. An analysis of the peri-implant tissue around bare expanded polytetrafluoroethylene (ePTFE), ePTFE embedded within a microvascular construct, or ePTFE embedded within collagen alone revealed that the presence of the MVC, but not collagen alone, promoted vascular densities comparable to that of the granulation tissue formed around bare ePTFE. The vessels within the microvascular construct surrounding the ePTFE were perfusion competent, as determined by India ink perfusion casting, and extended into the interstices of the polymer. In contrast to bare ePTFE, the presence of the MVC or collagen alone significantly reduced the number of activated macrophages in association with ePTFE. Similar results were observed for ePTFE modified to increase cellularity and prevent the formation of an avascular scar. The microvascular construct may prove effective in forming vascularized tissue environments and limiting the number of activated macrophages around implanted polymers thereby leading to effective implant incorporation.
granulation tissue; fibrous capsule; structural adaptation; microvascular networks
In the adult, angiogenesis leads to an expanded microvascular network as new vessel segments are added to an existing microcirculation. Necessarily, growing neovessels must navigate through tissue stroma as they locate and grow toward other vessel elements. We have a growing body of evidence demonstrating that angiogenic neovessels reciprocally interact with the interstitial matrix of the stroma resulting in directed neovascular growth during angiogenesis. Given the compliance and the viscoelastic properties of collagen, neovessel guidance by the stroma is likely due to compressive strain transverse to the direction of primary tensile forces present during active tissue deformation. Similar stromal strains control the final network topology of the new microcirculation, including the distribution of arterioles, capillaries, and venules. In this case, stromal-derived stimuli must be present during the post-angiogenesis remodeling and maturation phases of neovascularization to have this effect. Interestingly, the preexisting organization of vessels prior to the start of angiogenesis has no lasting influence on the final, new network architecture. Combined, the evidence describes interplay between angiogenic neovessels and stroma that is important in directed neovessel growth and invasion. This dynamic is also likely a mechanism by which global tissue forces influence vascular form and function.
angiogenesis; stroma; matrix; neovessel; remodeling
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.
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.
microcirculation; regeneration; bioprinting; vascular engineering; neovascularization
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.
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.
Vascular endothelial growth factor-A (VEGF) typically induces abnormal angiogenesis in the adult, thereby aggravating disease pathology and limiting utility of VEGF for therapeutic angiogenesis. To identify strategies for rectifying defects in pathological VEGF neovessels, we investigated consequences of modulating the Rho GTPase Cdc42. In a mouse skin model of VEGF-driven pathological angiogenesis, transduction with active Cdc42 (L28Cdc42) markedly improved VEGF neovessels, as measured by increased lumen formation, enlarged vessel diameter, and enhanced perfusion of macromolecular tracers. Conversely, transduction with dominant-negative Cdc42 (N17Cdc42) impaired endothelial cell (EC) assembly into lumenized blood vessels and reduced neovessel diameter and tracer perfusion. In vitro, active Cdc42 improved coordination between actin filaments and microtubules and enhanced formation of vascular cords, suggesting that active Cdc42 rectifies defects in angiogenesis by improving cytoskeletal dynamics and capillary morphogenesis. Analyses of Cdc42 signaling in microvascular ECs indicated that active Cdc42 also inhibits glycogen synthase kinase-3β (GSK-3β), a multi-functional serine/threonine protein kinase. Pharmacological inhibition of GSK-3β improved vascular cord formation in vitro and promoted proper neovessel formation in vivo comparably to active Cdc42, thus linking GSK-3β inhibition to the mechanism by which active Cdc42 rectifies pathological neovascularization. These studies identify activation of Cdc42 and inhibition of GSK-3β as novel strategies for correcting abnormalities associated with VEGF-driven angiogenesis, and they suggest new approaches for achieving improved therapeutic neovascularization with VEGF.
Vascular endothelial growth factor (VEGF); pathological angiogenesis; lumens; endothelial cells; cytoskeleton; Cdc42; glycogen synthase kinase-3β (GSK-3β); capillary morphogenesis
Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates.
In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN) calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT) calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs), increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks.
These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly. Accordingly, calpain represents an important target for rectifying key vascular defects associated with pathological angiogenesis and for improving therapeutic angiogenesis with VEGF.
The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and peri-vascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra-scaffold microvessel self-assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial- and vascular smooth muscle cells were seeded at different ratios in poly-L lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra-scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue engineering context was strongly enhanced in the implants seeded with a complete complement of blood vessel components: Human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel-enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra-scaffold microcirculation showed a uniform, branched microvascular network. 3-D image reconstruction analysis of hPASMC distribution within vascularized implants was non-random and displayed a preferential peri-vascular localization. Hence, efficient microvessel self-assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components.
angiogenesis; scaffold; endothelial; mural cell; microcirculation; multiphoton
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.
angiogenesis; microvessels; microvascular orientation; microvascular remodeling; microvessel guidance; three-dimensional (3D) vascular constructs; matrix mechanics
A novel tissue model for angiogenesis (TMA) is established for effective evaluation of angiogenesis inhibitors or promoters in vitro. Lung tissues were cultured in fibrinogen “sandwich” structure which resembled the formation of neovessels in vivo. The cells and capillary-like structures grew from the lung tissues were identified as endothelial cells and neovessels. Both immunohistochemisty and western blot results indicated that autocrine VEGF bound to the KDR and induced KDR autophosphorylation that could induce the proliferation of endothelial cells and their migration as well as the formation of microvessels on the lung tissue edge. With addition of the TMA, the murine VEGF and cultured medium produced by A549 tumor cells apparently promoted the increase of neovessels. Sorafenib as a tumor angiogenesis inhibitor and Tongxinluo as an angiogenesis promoter were both used to evaluate the TMA performance and they exhibited a good effect on neovessels in the TMA. The model established imitated angiogenesis in vivo and could well serve as an effective method in evaluating the angiogenesis inhibitors or promoters, and could also be practical for screening small molecules that affect blood vessel formation.
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.
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.
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.
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.
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.
Angiogenesis; vessel permeability; glial restricted precursors; astrocytes; angiogenesis assay
Successful therapeutic angiogenesis requires an understanding of how the milieu of growth factors available combine to form a mature vascular bed. This requires a model in which multiple physiological and cell biological parameters can be identified. The adenoviral-mediated mesenteric angiogenesis assay as described here is ideal for that purpose. Adenoviruses expressing growth factors (vascular endothelial growth factor [VEGF] and angiopoietin 1 [Ang-1]) were injected into the mesenteric fat pad of adult male Wistar rats. The clear, thin, and relatively avascular mesenteric panel was used to measure increased vessel perfusion by intravital microscopy. In addition, high-powered microvessel analysis was carried out by immunostaining of features essential for the study of angiogenesis (endothelium, pericyte, smooth muscle cell area, and proliferation), allowing functional data to be obtained in conjunction with high-power microvessel ultrastructural analysis. A combination of individual growth factors resulted in a distinct vascular phenotype from either factor alone, with all treatments increasing the functional vessel area. VEGF produced shorter, narrow, highly branched, and sprouting vessels with normal pericyte coverage. Ang-1 induced broader, longer neovessels with no apparent increase in branching or sprouting. However, Ang-1-induced blood vessels displayed a significantly higher pericyte ensheathment. Combined treatment resulted in higher perfusion, larger and less-branched vessels, with normal pericyte coverage, suggesting them to be more mature. This model can be used to show that Ang-1 and VEGF use different physiological mechanisms to enhance vascularisation of relatively avascular tissue.
Angiogenesis; angiopoietin 1; pericyte; VEGF
The purpose of this study was to define early events during the angiogenic response of the aortic wall to injury. Rat aortic rings produced neovessels in collagen culture but lost this capacity over time. These quiescent rings responded to vascular endothelial growth factor (VEGF) but not to a cocktail of macrophage-stimulatory cytokines and chemokines that was angiogenically active on fresh rings. Analysis of cytokine receptor expression revealed selective loss in quiescent rings of the proangiogenic chemokine receptor CXCR2, which was expressed predominantly in aortic macrophages. Pharmacologic inhibition of CXCR2 impaired angiogenesis from fresh rings but had no effect on VEGF-induced angiogenesis from quiescent explants. Angiogenesis was also impaired in cultures of aortic rings from CXCR2-deficient mice. Reduced CXCR2 expression in quiescent rat aortic rings correlated with marked macrophage depletion. Pharmacologic ablation of macrophages from aortic explants blocked formation of neovessels in vitro and reduced aortic ring-induced angiogenesis in vivo. The angiogenic response of macrophage-depleted rings was completely restored by adding exogenous macrophages. Moreover, angiogenesis from fresh rings was promoted by macrophage colony stimulating factor (CSF-1) and inhibited with anti-CSF-1 antibody. Thus aortic angiogenic sprouting following injury is strongly influenced by conditions that modulate resident macrophage numbers and function.
chemokines; inflammation; leukocytes; monocytes; neovascularization
One critical obstacle facing tissue engineering is the formation of functional vascular networks that can support tissue survival in vivo. We hypothesized that prevascularizing a tissue construct with networks of well-formed capillaries would accelerate functional anastomosis with the host upon implantation. Fibrin-based tissues were prevascularized with capillary networks by coculturing human umbilical vein endothelial cells (HUVECs) and fibroblasts in fibrin gels for 1 week. The prevascularized tissue and nonprevascularized controls were implanted subcutaneously onto the dorsal surface of immune-deficient mice and retrieved at days 3, 5, 7 and 14. HUVEC-lined vessels containing red blood cells were evident in the prevascularized tissue by day 5, significantly earlier than nonprevascularized tissues (14 days). Analysis of the HUVEC-lined vessels demonstrated that the number and area of perfused lumens in the prevascularized tissue were significantly larger compared to controls. In addition, collagen deposition and a larger number of proliferating cells were evident in the prevascularized tissue at day 14. Our results demonstrate that prevascularizing a fibrin-based tissue with well-formed capillaries accelerates anastomosis with the host vasculature, and promotes cellular activity consistent with tissue remodeling. Our prevascularization strategy may be useful to design large three-dimensional engineered tissues.
Dormant solid tumors were produced in vivo by prevention of neovascularization. When small fragments of anaplastic Brown-Pearce carcinoma were implanted directly on the iris in susceptible rabbits, they always vascularized. A characteristic growth pattern, consisting of prevascular, vascular, and late phases, was observed, which terminated with destruction of the eye within 2 wk. The beginning of exponential volume increase was shown to coincide with vascularization of the implant, as demonstrated by perfusion with intravenous fluorescein and by histologic sections. In contrast, implants placed in the anterior chamber, at a distance from the iris, did not become vascularized. After initial growth into spheroids, they remained arrested at a small size comparable to prevascular iris implants, for periods as long as 6 wk. Although dormant in terms of expansion, these avascular tumors contained a population of viable and mitotically active tumor cells. When reimplanted on the iris, vascularization was followed by rapid, invasive growth. These observations suggest that neovascularization is a necessary condition for malignant growth of a solid tumor. When a small mass of tumor cells is prevented from eliciting new vessel ingrowth from surrounding host tissues, population dormancy results. These data suggest that the specific blockade of tumor-induced angiogenesis may be an effective means of controlling neoplastic growth.
Engineering three-dimensional (3D) vascularized constructs remains a challenge due to the inability to form rich microvessel networks. In this study we engineered a prevascularized 3D cell sheet construct for tissue regeneration using human bone marrow-derived mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells as cell sources. hMSCs were cultured to form a thick cell sheet, and human umbilical vein endothelial cells (HUVECs) were then seeded on the hMSCs sheet to form networks. The single prevascularized HUVEC/hMSC cell sheet was folded to form a 3D construct by a modified cell sheet engineering technique. In vitro results indicated that the hMSCs cell sheet promoted the HUVECs cell migration to form networks in horizontal and vertical directions. In vivo results showed that many blood vessels grew into the 3D HUVEC/hMSC cell sheet constructs after implanted in the subcutaneous pocket of immunodeficient mice. The density of blood vessels in the prevascularized constructs was higher than that in the nonprevascularized constructs. Immunohistochemistry staining further showed that in vitro preformed human capillaries in the prevascularized constructs anastomosed with the host vasculature to form functional blood vessels. These results suggest the promising potential of this 3D prevascularized construct using hMSCs cell sheet as a platform for wide applications in engineering vascularized tissues.
Hyperpermeable tumor vessels are responsible for elevated interstitial fluid pressure and altered flow patterns within the tumor microenvironment. These aberrant hydrodynamic stresses may enhance tumor development by stimulating the angiogenic activity of endothelial cells lining the tumor vasculature. However, it is currently not known to what extent shear forces affect endothelial organization or paracrine signaling during tumor angiogenesis. The objective of this study was to develop a three-dimensional (3D), in vitro microfluidic tumor vascular model for coculture of tumor and endothelial cells under varying flow shear stress conditions. A central microchannel embedded within a collagen hydrogel functions as a single neovessel through which tumor-relevant hydrodynamic stresses are introduced and quantified using microparticle image velocimetry (μ-PIV). This is the first use of μ-PIV in a tumor representative, 3D collagen matrix comprised of cylindrical microchannels, rather than planar geometries, to experimentally measure flow velocity and shear stress. Results demonstrate that endothelial cells develop a confluent endothelium on the microchannel lumen that maintains integrity under physiological flow shear stresses. Furthermore, this system provides downstream molecular analysis capability, as demonstrated by quantitative RT-PCR, in which, tumor cells significantly increase expression of proangiogenic genes in response to coculture with endothelial cells under low flow conditions. This work demonstrates that the microfluidic in vitro cell culture model can withstand a range of physiological flow rates and permit quantitative measurement of wall shear stress at the fluid–collagen interface using μ-PIV optical flow diagnostics, ultimately serving as a versatile platform for elucidating the role of fluid forces on tumor–endothelial cross talk.
Plaque angiogenesis may have an important role in the development of atherosclerosis. Vasa vasorum angiogenesis and medial infiltration provides nutrients to the developing and expanding intima and therefore, may prevent cellular death and contribute to plaque growth and stabilization in early lesions. However in more advanced plaques, inflammatory cell infiltration, and concomitant production of numerous pro-angiogenic cytokines may be responsible for induction of uncontrolled neointimal microvessel proliferation resulting in production of immature and fragile neovessels similar to that seen in tumour development. These could contribute to development of an unstable haemorrhagic rupture-prone environment. Increasing evidence has suggested that the expression of intimal neovessels is directly related to the stage of plaque development, the risk of plaque rupture, and subsequently, the presence of symptomatic disease, the timing of ischemic neurological events and myocardial/cerebral infarction. Despite this, there is conflicting evidence regarding the causal relationship between neovessel expression and plaque thrombosis with some in vivo experimental models suggesting the contrary and as yet, few direct mediators of angiogenesis have been identified and associated with plaque instability in vivo.
In recent years, an increasing number of angiogenic therapeutic targets have been proposed in order to facilitate modulation of neovascularization and its consequences in diseases such as cancer and macular degeneration. A complete knowledge of the mechanisms responsible for initiation of adventitial vessel proliferation, their extension into the intimal regions and possible de-novo synthesis of neovessels following differentiation of bone-marrow-derived stem cells is required in order to contemplate potential single or combinational anti-angiogenic therapies. In this review, we will examine the importance of angiogenesis in complicated plaque development, describe the current knowledge of molecular mechanisms of its initiation and maintenance, and discuss possible future anti-angiogenic therapies to control plaque stability.
To ensure the survival of engineered bone after implantation, we combined human endothelial colony forming cells (ECFCs) and multipotent stromal cells (MSCs) as a proof of concept in a co-culture model to create in vitro prevascularized bone constructs. We hypothesized that a hypoxic stimulus will contribute to prevascularization of engineered bone. Bone marrow-derived MSCs and ECFCs from human adult peripheral blood were allowed to form co-culture pellets containing ECFCs and MSCs (1:4) or MSCs only in controls. After culture under normoxia or hypoxia (5%), pellets were harvested and processed for immunohistochemistry of CD31, α-smooth muscle actin, and osteocalcin. Expression of vascular endothelial growth factor and SDF-1α was analyzed by PCR to elucidate their involvement in hypoxic stimulation of prevascularization. The normoxic condition in co-cultures of MSCs and ECFCs supported the formation and maintenance of prevascular structures, including organized CD31-positive cells embraced by differentiated mural cells. These structures failed to form in hypoxic conditions, thereby rejecting the hypothesis that hypoxia stimulates prevasculogenesis in three-dimensional engineered bone constructs. Further, the formation of prevascular structures was paralleled by increased SDF-1α expression. It is suggested that actual oxygen levels were below 5% in the hypoxic co-cultures, which prevented prevascular structure formation. In conclusion, our normoxic co-culture model containing cells from clinically relevant sources sustained simultaneous endothelial, smooth muscle, and osteogenic differentiation.
Photodynamic therapy (PDT) induces damage to the endothelium, which can lead to increased vascular permeability and, under intensive PDT conditions, even to platelet aggregation, vasoconstriction, and blood flow stasis. Eventually, ischemia, hypoxia, and inflammation can occur, resulting in angiogenesis. We studied the sequence of the vascular events after Visudyne®-PDT in the chicken chorioallantoic membrane (CAM) at day 11 of development. Using epi-fluorescence microscopy, we monitored the regrowth of capillaries in the PDT treated area. Immediately after irradiation, the treatment resulted in blood flow arrest. And 24 h post PDT, sprouting of new blood vessels was observed at the edge of the PDT zone. Neovessels looping out from the edge of the PDT zone gave rise to specialized endothelial tip structures guiding the vessels towards the center of the treated area. At 48 h almost all of the treated area was repopulated with functional but morphologically altered vasculature. These observations also showed reperfusion of some of the vessels that had been closed by the PDT treatment. CAM samples were immunohistochemically stained for Ki-67 showing proliferation of endothelial cells in the PDT area. Also, several markers of immature and angiogenic blood vessels, such as αVβ3-integrin, vimentin and galectin-1, were found to be enhanced in the PDT area, while the endothelial maturation marker intercellular adhesion molecule (ICAM)-1 was found to be suppressed. These results demonstrate that the new vascular bed is formed by both neo-angiogenesis and reperfusion of existing vessels. Both the quantitative real-time RT–PCR profile and the response to pharmacological treatment with Avastin®, an inhibitor of angiogenesis, suggest that angiogenesis occurs after PDT. The observed molecular profiling results and the kinetics of gene regulation may enable optimizing combination therapies involving PDT for treatment of cancer and other diseases.
Photodynamic therapy; Capillary; VEGF; Angiogenesis; Real-time RT–PCR; Visudyne®; Avastin®; Galectin-1; Vimentin
Formation of haemorrhagic neovessels in the intima of developing atherosclerotic plaques is thought to significantly contribute to plaque instability resulting in thrombosis. C-reactive protein (CRP) is an acute phase reactant whose expression in the vascular wall, in particular, in reactive plaque regions, and circulating levels increase in patients at high risk of cardiovascular events. Although CRP is known to induce a pro-inflammatory phenotype in endothelial cells (EC) a direct role on modulation of angiogenesis has not been established.
Here, we show that CRP is a powerful inducer of angiogenesis in bovine aortic EC (BAEC) and human coronary artery EC (HCAEC). CRP, at concentrations corresponding to moderate/high risk (1–5 μg/ml), induced a significant increase in proliferation, migration and tube-like structure formation in vitro and stimulated blood vessel formation in the chick chorioallantoic membrane assay (CAM). CRP treated with detoxi-gel columns retained such effects. Western blotting showed that CRP increased activation of early response kinase-1/2 (ERK1/2), a key protein involved in EC mitogenesis. Furthermore, using TaqMan Low-density Arrays we identified key pro-angiogenic genes induced by CRP among them were vascular endothelial cell growth factor receptor-2 (VEGFR2/KDR), platelet-derived growth factor (PDGF-BB), notch family transcription factors (Notch1 and Notch3), cysteine-rich angiogenic inducer 61 (CYR61/CCN1) and inhibitor of DNA binding/differentiation-1 (ID1).
This data suggests a role for CRP in direct stimulation of angiogenesis and therefore may be a mediator of neovessel formation in the intima of vulnerable plaques.
Bone marrow-derived endothelial progenitor cells (EPCs) contribute to angiogenesis-mediated pathological neovascularization and recent studies have begun to recognize the biological significance of this contribution. This review will discuss the ability of EPCs to contribute to neovascularization in both physiological and pathological conditions. Circulating EPCs were originally identified in 1997 by Asahara as CD34+ VEGFR2+ mononuclear cells. These cells differentiated into an endothelial phenotype, expressed endothelial markers, and incorporated into neovessels at sites of ischemia (Asahara et al., 1997). EPCs provide both instructive (release of pro-angiogenic cytokines) and structural (vessel incorporation and stabilization) functions that contribute to the initiation of neo-angiogenesis. EPC populations can be characterized based on surface markers of freshly isolated cells or they can be described by their in vitro characteristics once placed in culture. However, a major stumbling block to progress in the field has been the lack of consensus among investigators as to the optimal characterization of EPCs. This review intends to address the role of both EPC classes and evaluate how they interact in the setting of pathological angiogenesis. Since the EPCs may be responsible for turning on the “angiogenic switch,” strategies have been employed to keep this switch in the “off” position for diseases like cancer, retinopathy and wet AMD. The expectation is that EPCs will evolve into clinically useful prognostic and predictive tools in cancer and in ocular diseases associated with pathological neovascularization and that targeting this cell type is a key to successful management of patients suffering from diseases associated with pathological neovascularization.
Previous studies have demonstrated that expression of CD105 is a sensitive marker and indicator of endothelial cell/microvessel activation and proliferation in aggressive solid tumour growth and atherosclerotic plaque lesions. Since intimal neovascularization contributes significantly to subsequent plaque instability, haemorrhage and rupture.
We have used immunohistochemical analysis to investigate the expression of CD105-positive vessels in both large (carotid) and medium calibre (coronary and middle cerebral artery, MCAs) diseased vessels in an attempt to identify any correlation with plaque growth, stage and complication/type.
Here we show, that carotid arteries expressed intimal neovascularization associated with CD105-positive endothelial cells, concomitant with increased inflammation in early stage lesions, preatheroma (I-III) whilst they were not present in coronary plaques of the same grade. Some of these CD105-positive neovessels were immature, thin walled and without smooth muscle cell coverage making them more prone to haemorrhage and rupture. In high-grade lesions, neovessel proliferation was similar in both arterial types and significantly higher numbers of CD105-positive vasa vasorum were associated with plaque regions in coronary arteries. In contrast, although the MCAs exhibited expanded intimas and established plaques, there were very few CD105 positive neovessels.
Our results show that CD105 is a useful marker of angiogenesis within adventitial and intimal vessels and suggest the existence of significant differences in the pathological development of atherosclerosis in separate vascular beds which may have important consequences when considering management and treatment of this disease.