Diane C. Darland

Engineering vascularized skeletal muscle tissue

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.

Endogenous VEGF Is Required for Visual Function: Evidence for a Survival Role on Müller Cells and Photoreceptors

Background Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical. Methods and Findings Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective. Conclusions These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.

Blood vessel maturation: vascular development comes of age

What are the cellular and molecular mechanisms that regulate new vessel formation and maturation? This question identifies several fundamental issues of vascular biology related to the development of the vasculature via vasculogenesis and angiogenesis. Early investigations of vasculature were largely descriptive (1, 2). The advent of methods for the isolation and maintenance of vascular cells in tissue culture led to assay systems aimed at recreating the initial events of the angiogenic process, including endothelial cell protease production, migration and proliferation. However, less attention has been directed to the later stages of vascularization, such as investment of vessels with mural cells (pericytes or smooth muscle cells), production of basement membrane, induction of vessel bed specializations or the process of vascular regression. The final stages of vessel formation might also be referred to as “remodeling” and appear to define a process through which a forming vasculature becomes a stable, mature vessel bed. These steps may also be taken advantage of to develop antitumor strategies, as suggested by Benjamin et al. in this issue (3). What regulates the phenotypic changes that occur between forming and mature vessels? A number of clinical and experimental observations support the concept that the association between the vascular tube and the mural cells mediates vessel stabilization or maturation. Ultrastructural studies of vascularization during wound healing revealed a temporal correlation between pericyte investment of the capillary and the cessation of vessel growth. This observation led the authors of this study to state: “The incorporation of pericytes within the basement membrane of proliferating capillaries is proposed as the mechanism for inhibition of capillary proliferation” (4). Clinical observations are consistent with a role for mural cells in stabilizing vessels. The vessel proliferation that characterizes proliferative diabetic retinopathy is preceded by a stage characterized by the selective loss of pericytes (5). Similarly, the new vessels that form as a complication of the retinopathy of prematurity arise from immature microvessels that have yet to be invested by pericytes (6). Thus, the physical association between the nascent vascular tube and mural cells appears to signal vessel stabilization. Earlier work from Stone and Keshet indicates that vessels are dependent upon exogenous survival factors, e.g., VEGF (7) (Fig. ​(Fig.1),1), for a critical period of time during their development. The current study by Benjamin et al. in this issue (3) further suggests that the association of forming vessels with the mural cells marks the end of this period of growth factor dependence. Two aspects of vessel stabilization are addressed in this study. The first is that in the absence of associated pericytes or smooth muscle cells, the nascent endothelial cell tube is unstable and is prone to regression. The second is the notion that the nascent endothelial tube requires VEGF for survival. In normal development, the time for overlap of these events is terminated by the arrival of the mural cells. The tumor vasculature, on the other hand, appears to be trapped in a cycle where high levels of growth factors can induce and sustain immature vessels in the relative absence of mural cells. The chronic immaturity of tumor vessels has led Dvorak to characterize a tumor as a “wound that never heals” (8). At the same time, these features make tumor vessels viable targets for antitumor therapies. Benjamin et al. (3) demonstrate that removal of growth factors leads not only to the cessation of new vessel growth, but also to regression of the immature tumor vasculature (3). Figure 1 Vessel maturation: vessel development proceeds from a stage of growth-factor dependence where loss of a survival factor leads to apoptosis. Vessel stabilization is marked by investment with mural cells, local activation of TGF-β, and basement ... Although recent evidence suggests that endothelial cell–mural cell contact and VEGF-dependence are pivotal junctures during vessel maturation, questions regarding the molecular basis of vessel maturation remains unanswered. Using tissue culture models, we have previously shown that endothelial cells recruit mural cell precursors via the secretion of PDGF-BB. Consistent with this prediction, mice null for PDGF B lack pericytes in some microvascular beds (10). Furthermore, coculture studies reveal that endothelial cell–mural cell contact leads to the activation of TGF-β (9, 11), a pleiotrophic growth factor that has been shown to mediate differentiation of a variety of cell types. In the context of the microvasculature, we speculate that TGF-β functions at multiple steps leading to vessel stabilization, including inhibition of endothelial cell proliferation and migration, as well as induction of pericyte/smooth muscle cell differentiation (12) (Fig. ​(Fig.1).1). Once tube formation occurs, flow commences and local oxygen levels increase, resulting in a decrease in VEGF levels. The reduction in VEGF is coincident with pericyte/smooth muscle cell association. In addition, TGF–β can alter integrin profiles and stimulate basement membrane production and accumulation (for review) (13). In support of this idea, ultrastructural studies document that a basement membrane is deposited only after endothelial cell–mural cell association has occurred (4). Thus, the presence of basement membrane may provide a long-term signal that supports vessel stabilization. Furthermore, recent evidence suggests that the angiopoietins play a role in vessel stabilization (for review) (14). Angiopoietin-1 is associated with developing vessels and its absence leads to defects in vessel remodeling. On the other hand, angiopoietin-2, which antagonizes the actions of angiopoietin-1, appears to play a role in the destabilization of existing vessels. Although the precise role played by each of these factors is not clear, their existence suggests yet another level of control during vessel maturation (Fig. ​(Fig.1).1). Thus, vessel stabilization results from a balance between stimulators (such as VEGF) and inhibitors (such as TGF-β), all acting in the context of the vessel microenvironment. The idea that cell–cell interactions mediate differentiation and stabilization of developing tissues is not restricted to the vasculature. Similar interactions have been described in the developing kidney (15), lung (16), mammary gland (17), and heart (18) where there are heterotypic interactions between epithelial and mesenchymal cells, associated in many cases, with the local production of members of the TGF-β superfamily. An additional parallel can be drawn to the developing nervous system, where heterotypic interactions between an innervating neuron and its target cell effect the survival and/or differentiation of both cell types (19). Thus, heterotypic cell–cell interactions may be a universal mechanism for the local regulation of cell-type specification and differentiation. Whereas the earliest studies of the vasculature described cell behavior in situ, and the past two decades have focused on dissecting the function of isolated vascular cells in culture, we are now at a transition point which recognizes the complexity of the microvasculature (20). Most importantly, the importance of heterotypic cell–cell interactions, local control of growth factor expression (e.g., hypoxic regulation of VEGF, local activation of TGF-β), and the signaling potential of the basement membrane are acknowledged and being investigated. As a result, new questions continue to arise. Is vessel assembly controlled similarly in all vascular beds? Studies involving PDGF B-deficient mice suggest there are tissue-specific differences, since not all microvascular beds in these mice lacked pericytes. What is the relevance of the fact that different microvessel beds have different levels of coverage by pericytes? Many prostatic vessels have few pericytes, retinal and brain microvessels have extensive coverage, lymphatic vessels have no mural cells at all, and pericytes presence in tumor vessels is variable. What is the nature of the heterotypic cell–cell interactions between endothelial cells and mural cells? Is there a role for gap junctions and/or signaling via adhesion molecules? Which diffusible factors are involved in this communication (e.g., angiopoietins, VEGFs, PDGFs, TGF-family members, etc.)? What is the role of growth factors in the adult vasculature? Why is there continued production of VEGF in adult tissues if mature vessels are VEGF-independent? Are there other survival factors? Does TGF-β play a role in vessel maturation? What is the role of the basement membrane? Understanding the cellular and molecular basis of vessel maturation is central to delineating the full “life cycle” of blood vessels and lays the groundwork for developing effective pro-angiogenic and anti-angiogenic therapies.

TGFβ is required for the formation of capillary-like structures in three-dimensional cocultures of 10T1/2 and endothelial cells

New vessels form de novo (vasculogenesis) or from pre-existing vessels (angiogenesis) in a process that involves the interaction of endothelial cells (EC) and pericytes/smooth muscle cells (SMC). One basic component of this interaction is the endothelial-induced recruitment, proliferation and subsequent differentiation of pericytes and SMC. We have previously demonstrated that TGFβ induces the differentiation of C3H/10T1/2 (10T1/2) mesenchymal cells toward a SMC/pericyte lineage. The current study tests the hypothesis that TGFβ not only induces SMC differentiation but stabilizes capillary-like structures in a three-dimensional (3D) model of in vitro angiogenesis. 10T1/2 and EC in Matrigel™ were used to establish cocultures that form cord structures that are reminiscent of new capillaries in vivo. Cord formation is initiated within 2–3 h after plating and continues through 18 h after plating. In longer cocultures the cord structures disassemble and form aggregates. 10T1/2 expression of proteins associated with the SMC/pericyte lineage, such as smooth muscle α-actin (SMA) and NG2 proteoglycan, are upregulated in these 3D cocultures. Application of neutralizing reagents specific for TGFβ blocks cord formation and inhibits expression of SMA and NG2 in the 10T1/2 cells. We conclude that TGFβ mediates 10T1/2 differentiation to SMC/pericytes in the 3D cocultures and that association with differentiated mural cells is required for formation of capillary-like structures in Matrigel™.

Cell cell interactions in vascular development

Research into areas as divergent as hemangiopoiesis and cardiogenesis as well as investigations of diseases such as cancer and diabetic retinopathy have converged to form the face of research in vascular development today. This convergence of disparate topics has resulted in rapid advances in many areas of vascular research. The focus of this review has been the role of cell-cell interactions in the development of the vascular system, but we have included discussions of pathology where the mechanism of disease progression may have parallels with developmental processes. A number of intriguing questions remain unanswered. For example, what triggers abnormal angiogenesis in the disease state? Are the mechanisms similar to those that control developmental neovascularization? Perhaps the difference in development in angiogenesis versus in disease is context driven, that is, an adult versus an embryonic organism. If this is the case, can the controls that curtail developmental vessel formation be applied in pathologies? Can cell-cell interactions be targeted as a control point for new vessel formation? For instance, can perivascular cells be stimulated or eliminated to result in increased vessel stability or instability, respectively? If the hypothesis that mural cell association is required for vessel stabilization is accurate, are there mechanisms to promote or inhibit mural cell recruitment and differentiation as needed? These and other questions lie in wait for the next generation of approaches to discern the mechanisms and the nature of the cell-cell interactions and the influence of the microenvironment on vascular development.

Retinal pigment epithelium and endothelial cell interaction causes retinal pigment epithelial barrier dysfunction via a soluble VEGF-dependent mechanism

PURPOSE To investigate the effect of endothelial cells (EC) on the barrier function of the retinal pigment epithelium (RPE). METHODS Primary bovine RPE were grown in solo culture or in coculture with bovine EC. Culture media of RPE were varied to develop a monolayer with stable barrier properties determined by transepithelial electrical resistance (TER) and permeability to sodium fluorescein. The effect of EC on the barrier properties of RPE was tested in contacting and non-contacting cocultures of RPE and EC. The conditioned media of cocultures were analysed for soluble vascular endothelial growth factor (VEGF) by ELISA. A neutralizing antibody to VEGF(165) was added to cocultures of RPE and EC and the TER was measured. RESULTS RPE had maximal barrier properties (high TER, low permeability, positive staining for barrier proteins) at day 10 that persisted until day 20. Compared to solo RPE culture, cocultivation of RPE with EC reduced RPE barrier function significantly and led to a greater release of soluble VEGF into the conditioned media (p<0.05). Neutralizing VEGF with antibody led to partial recovery of barrier properties in the coculture conditions (p<0.03). CONCLUSIONS Coculture of RPE with EC reduces RPE barrier properties and the reduction is, in part, mediated by soluble VEGF. EC-RPE contact-induced disruption of barrier properties occurs in ocular pathologies such as choroidal neovascularization, where EC move through Bruchs membrane and contact the RPE, leading to further exacerbation of the already compromised blood-retinal barrier.

Functional analysis of a mutant form of the receptor tyrosine kinase Tie2 causing venous malformations

Tie2 is expressed predominantly in endothelial cells and is required for blood vessel formation and maintenance. A missense mutation resulting in an R to W substitution in the kinase domain of Tie2 co-segregates with an autosomal dominantly inherited form of vascular dysmorphogenesis, venous malformation (VM). The mechanism by which this activating mutation leads to vessel dysmorphogenesis in VM is not known. Here we examined Tie2 activation status in VM and found activated receptor in lesional and non-lesional vessels. To gain insight into functional effects of VM mutant Tie2, wild-type and R849W mutant receptor were expressed in cultured human venous endothelial cells. Mutant Tie2 was constitutively phosphorylated in endothelial cells in vivo and caused a marked suppression of apoptosis. The anti-apoptotic kinase Akt was constitutively activated in cells expressing mutant receptor. Dominant-negative Akt inhibited the pro-survival activity of mutant Tie2. Migration of smooth muscle cells induced by conditioned medium from cells expressing mutant receptor was similar to that from cells expressing wild-type receptor. These data suggest that a primary effect of R849W Tie2 in VM is to allow survival of mural cell poor vessels via ligand-independent Tie2 activation of Akt and endothelial survival, rather than to directly induce formation of dysmorphogenic vessels.

In vivo adenovirus-mediated delivery of a uPA/uPAR antagonist reduces retinal neovascularization in a mouse model of retinopathy.

Diabetic retinopathy and retinopathy of prematurity are among the leading causes of vision impairment throughout the world. Both diseases are characterized by pathological angiogenesis, which severely impairs vision. Extracellular proteinases play important roles in endothelial cell migration during angiogenesis. Amino-terminal fragment (ATF) is an angiostatic molecule that targets the uPA/uPAR system and inhibits endothelial cell migration. The angiostatic effect of ATF has been demonstrated in models of cancer, but has never been assessed in pathological retinal neovascularization. Endostatin also has angiostatic effects on tumor growth and retinal neovascularization.We used an adenoviral vector carrying the murine ATF (AdATFHSA) or endostatin gene coupled to human serum albumin (HSA) (AdEndoHSA) to increase the half-life of the therapeutic protein in the circulation. We induced retinopathy by exposing 7-day-old mice to high levels of oxygen. They were intravitreally injected with the vectors. Local injection of AdATFHSA or AdEndoHSA reduced retinal neovascularization by 78.1 and 79.2%, respectively. Thus, the adenovirus-mediated delivery of ATFHSA or EndoHSA reduces retinal neovascularization in a mouse model of hypoxia-induced neovascularization.

Vascular endothelial growth factor (VEGF) isoform regulation of early forebrain development

This work was designed to determine the role of the vascular endothelial growth factor A (VEGF) isoforms during early neuroepithelial development in the mammalian central nervous system (CNS), specifically in the forebrain. An emerging model of interdependence between neural and vascular systems includes VEGF, with its dual roles as a potent angiogenesis factor and neural regulator. Although a number of studies have implicated VEGF in CNS development, little is known about the role that the different VEGF isoforms play in early neurogenesis. We used a mouse model of disrupted VEGF isoform expression that eliminates the predominant brain isoform, VEGF164, and expresses only the diffusible form, VEGF120. We tested the hypothesis that VEGF164 plays a key role in controlling neural precursor populations in developing cortex. We used microarray analysis to compare gene expression differences between wild type and VEGF120 mice at E9.5, the primitive stem cell stage of the neuroepithelium. We quantified changes in PHH3-positive nuclei, neural stem cell markers (Pax6 and nestin) and the Tbr2-positive intermediate progenitors at E11.5 when the neural precursor population is expanding rapidly. Absence of VEGF164 (and VEGF188) leads to reduced proliferation without an apparent effect on the number of Tbr2-positive cells. There is a corresponding reduction in the number of mitotic spindles that are oriented parallel to the ventricular surface relative to those with a vertical or oblique angle. These results support a role for the VEGF isoforms in supporting the neural precursor population of the early neuroepithelium.

Sulpiride, but not SCH23390, modifies cocaine-induced conditioned place preference and expression of tyrosine hydroxylase and elongation factor 1α in zebrafish

Finding genetic polymorphisms and mutations linked to addictive behavior can provide important targets for pharmaceutical and therapeutic interventions. Forward genetic approaches in model organisms such as zebrafish provide a potentially powerful avenue for finding new target genes. In order to validate this use of zebrafish, the molecular nature of its reward system must be characterized. We have previously reported the use of cocaine-induced conditioned place preference (CPP) as a reliable method for screening mutagenized fish for defects in the reward pathway. Here we test if CPP in zebrafish involves the dopaminergic system by co-treating fish with cocaine and dopaminergic antagonists. Sulpiride, a potent D2 receptor (DR2) antagonist, blocked cocaine-induced CPP, while the D1 receptor (DR1) antagonist SCH23390 had no effect. Acute cocaine exposure also induced a rise in the expression of tyrosine hydroxylase (TH), an important enzyme in dopamine synthesis, and a significant decrease in the expression of elongation factor 1α (EF1α), a housekeeping gene that regulates protein synthesis. Cocaine selectively increased the ratio of TH/EF1α in the telencephalon, but not in other brain regions. The cocaine-induced change in TH/EF1α was blocked by co-treatment with sulpiride, but not SCH23390, correlating closely with the action of these drugs on the CPP behavioral response. Immunohistochemical analysis revealed that the drop in EF1α was selective for the dorsal nucleus of the ventral telencephalic area (Vd), a region believed to be the teleost equivalent of the striatum. Examination of TH mRNA and EF1α transcripts suggests that regulation of expression is post-transcriptional, but this requires further examination. These results highlight important similarities and differences between zebrafish and more traditional mammalian model organisms.