Modulation of microvascular growth and morphogenesis by reconstituted basement membrane gel in three-dimensional cultures of rat aorta: A comparative study of angiogenesis in Matrigel,collagen, fibrin,and plasma clot

Summary Rings of rat aorta cultured in Matrigel, a reconstituted gel composed of basement membrane molecules, gave rise to three-dimensional networks composed of solid cellular cords and occasional microvessels with slitlike lumina. Immunohistochemical and ultrastructural studies showed that the solid cords were composed of endothelial sprouts surrounded by nonendothelial mesenchymal cells. The angiogenic response of the aortic rings in Matrigel was compared to that obtained in interstitial collagen, fibrin, or plasma clot. Morphometric analysis demonstrated that the mean luminal area of the microvascular sprouts and channels was significantly smaller in Matrigel than in collagen, fibrin, or plasma clot. The percentage of patent microvessels in Matrigel was also markedly reduced. Autoradiographic studies of³H-thymidine-labeled cultures showed reduced DNA synthesis by developing microvessels in Matrigel. The overall number of solid endothelial cords and microvessels was lower in Matrigel than in fibrin or plasma clot. A mixed cell population isolated from Matrigel cultures formed a monolayer in collagen or fibrin-coated dishes but rapidly reorganized into a polygonal network when plated on Matrigel. The observation that gels composed of basement membrane molecules modulate the canalization, proliferation, and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that the basement membrane is a potent regulator of microvascular growth and morphogenesis. This work was supported by grants from the W. W. Smith Charitable Trust and grants CA14137 and HL43392 from the National Institutes of Health, Bethesda, MD.     

Pro-angiogenic signaling by the endothelial presence of CEACAM1

Here, we demonstrate the expression of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) in angiogenic sprouts but not in large mother blood vessels within tumor tissue. Correspondingly, only human microvascular endothelial cells involved in in vitro tube formation exhibit CEACAM1. CEACAM1-overexpressing versus CEACAM1-silenced human microvascular endothelial cells were used in migration and tube formation assays. CEACAM1-overexpressing microvascular endothelial cells showed prolonged survival and increased tube formation when they were stimulated with vascular endothelial growth factor (VEGF), whereas CEACAM1 silencing via small interfering RNA blocks these effects. Gene array and LightCycler analyses show an up-regulation of angiogenic factors such as VEGF, VEGF receptor 2, angiopoietin-1, angiopoietin-2, tie-2, angiogenin, and interleukin-8 but a down-regulation of collagen XVIII/endostatin and Tie-1 in CEACAM1-overexpressing microvascular endothelial cells. Western blot analyses confirm these results for VEGF and endostatin at the protein level. These results suggest that constitutive expression of CEACAM1 in microvascular endothelial cells switches them to an angiogenic phenotype, whereas CEACAM1 silencing apparently abrogates the VEGF-induced morphogenetic effects during capillary formation. Thus, strategies targeting the endothelial up-regulation of CEACAM1 might be promising for antiangiogenic tumor therapy.     

CEA-related cell adhesion molecule 1: a potent angiogenic factor and a major effector of vascular endothelial growth factor

CEA-related cell adhesion molecule 1 (CEACAM1) exhibits angiogenic properties in in vitro and in vivo angiogenesis assays. CEACAM1 purified from granulocytes and endothelial cell media as well as recombinant CEACAM1 expressed in HEK293 cells stimulate proliferation, chemotaxis, and capillary-like tube formation of human microvascular endothelial cells. They increase vascularization of chick chorioallantoic membrane and potentiate the effects of vascular endothelial growth factor (VEGF)165. VEGF165 increases CEACAM1 expression both on the mRNA and the protein level. VEGF165-induced endothelial tube formation is blocked by a monoclonal CEACAM1 antibody. These data suggest that CEACAM1 is a major effector of VEGF in the early microvessel formation. Since CEACAM1 is expressed in tumor microvessels but not in large blood vessels, CEACAM1 may be a target for the inhibition of tumor angiogenesis.     

Recent progress in understanding Bartonella-induced vascular proliferation

The ability to induce endothelial cell proliferation is a common feature of human pathogenic Bartonella species. Recent data have indicated that bartonellae can provoke angioproliferation by at least two independent mechanisms: directly, by triggering proliferation and inhibiting apoptosis of endothelial cells; and indirectly, by stimulating a paracrine angiogenic loop of vascular endothelial growth factor production by infected macrophages. A NF-kappaB-mediated acute inflammatory reaction of the Bartonella-infected endothelium appears to be critical for the recruitment of monocytes/macrophages and the initiation and maintenance of a paracrine angiogenic loop. Given that bartonellae effectively adhere to and invade endothelial cells, their ability to trigger angioproliferation might represent a dedicated pathogenic strategy for expanding the bacterial host cell habitat.     

Paracrine regulation of angiogenesis by different cell types in the aorta ring model

The development of blood vessels during angiogenesis is the result of paracrine interactions between tube-forming endothelial cells and angiogenic factor-producing nonendothelial cells. This process can be reproduced and studied under chemically defined culture conditions by culturing vascular explants in three-dimensional gels of extracellular matrix. Rings of rat or mouse aorta cultured in collagen, fibrin or basement membrane gels produce angiogenic outgrowths composed of a mixed population of endothelial cells and nonendothelial cells. Aortic angiogenesis is regulated by endogenous angiogenic factors, inflammatory cytokines, chemokines, extracellular matrix molecules, and proteolytic enzymes produced by cells of the vessel wall in response to the injury of the dissection procedure. In this paper, we review how macrophages, mural cells and fibroblasts regulate different stages of the angiogenic process, from the formation of immature endothelial sprouts to the reabsorption of the neovessels. We also describe how aortic cultures can be used to study interactions between angiogenic outgrowths and nonvascular cell types such as bone marrow macrophages, platelets or cancer cells. Morphologic, genetic and functional studies of this model have provided invaluable information on how vessels form, mature, interact with nonvascular cell types, and are eventually reabsorbed. Further analysis of the paracrine cross-talk between aortic endothelial and nonendothelial cells is likely to provide new insights into the angiogenic process and its key mechanisms.     

Differentiation state determines neural effects on microvascular endothelial cells

Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells.     

Flow shear stress regulates endothelial barrier function and expression of angiogenic factors in a 3D microfluidic tumor vascular model

Endothelial cells lining blood vessels are exposed to various hemodynamic forces associated with blood flow. These include fluid shear, the tangential force derived from the friction of blood flowing across the luminal cell surface, tensile stress due to deformation of the vessel wall by transvascular flow, and normal stress caused by the hydrodynamic pressure differential across the vessel wall. While it is well known that these fluid forces induce changes in endothelial morphology, cytoskeletal remodeling, and altered gene expression, the effect of flow on endothelial organization within the context of the tumor microenvironment is largely unknown. Using a previously established microfluidic tumor vascular model, the objective of this study was to investigate the effect of normal (4 dyn/cm²), low (1 dyn/cm²), and high (10 dyn/cm²) microvascular wall shear stress (WSS) on tumor-endothelial paracrine signaling associated with angiogenesis. It is hypothesized that high WSS will alter the endothelial phenotype such that vascular permeability and tumor-expressed angiogenic factors are reduced. Results demonstrate that endothelial permeability decreases as a function of increasing WSS, while co-culture with tumor cells increases permeability relative to mono-cultures. This response is likely due to shear stress-mediated endothelial cell alignment and tumor-VEGF-induced permeability. In addition, gene expression analysis revealed that high WSS (10 dyn/cm²) significantly down-regulates tumor-expressed MMP9, HIF1, VEGFA, ANG1, and ANG2, all of which are important factors implicated in tumor angiogenesis. This result was not observed in tumor mono-cultures or static conditioned media experiments, suggesting a flow-mediated paracrine signaling mechanism exists with surrounding tumor cells that elicits a change in expression of angiogenic factors. Findings from this work have significant implications regarding low blood velocities commonly seen in the tumor vasculature, suggesting high shear stress-regulation of angiogenic activity is lacking in many vessels, thereby driving tumor angiogenesis.     

FGF2-dependent neovascularization of subcutaneous Matrigel plugs is initiated by bone marrow-derived pericytes and macrophages

Vessel-like networks are quickly formed in subcutaneous FGF2-supplemented Matrigel plugs by two cell types: NG2(+) pericytes and F4/80(+) macrophages. Although not detected in these networks until 7 days after plug implantation, the appearance of CD31(+) endothelial cells marks the onset of vessel perfusion and the establishment of mature vessel morphology, with endothelial cells invested tightly by pericytes and more loosely by macrophages. Evidence that mature vessels develop from pericyte/macrophage networks comes from experiments in which 5-day plugs are transplanted into EGFP(+) recipients and allowed to mature. Fewer than 5% of pericytes in mature vessels are EGFP(+) in this paradigm, demonstrating their presence in the networks prior to plug transplantation. Endothelial cells represent the major vascular cell type recruited during later stages of vessel maturation. Bone marrow transplantation using EGFP(+) donors establishes that almost all macrophages and more than half of the pericytes in Matrigel vessels are derived from the bone marrow. By contrast, only 10% of endothelial cells exhibit a bone marrow origin. The vasculogenic, rather than angiogenic, nature of this neovascularization process is unique in that it is initiated by pericyte and macrophage progenitors, with endothelial cell recruitment occurring as a later step in the maturation process.     

Studying the growth of cervical carcinoma nests and angiogenesis by immunostaining, quantitation and three-dimensional structural analysis

OBJECTIVE: To analyze the relationship and mutual effect of the growth of cervical carcinoma nests and angiogenesis. STUDY DESIGN: Serial paraffin sections of cervical squamous carcinoma were stained in repeated order with hematoxylin-eosin (HE), immunostain for factor VIII-related antigen, type IV collagen and proliferating cell nuclear antigen (PCNA). Three-dimensional reconstructions were made, and the volumes of carcinoma nests, necrosis and microvessels were measured. RESULTS: Two types of cervical carcinoma nests were distinguished on the basis of their growth characteristics and vascularity (groups I and II). Group I nests: The carcinoma cells were proliferating actively, as determined by their morphology and PCNA staining. There were abundant microvessels. Some endothelial sprouts and cords penetrated the nests and then formed networks and new vessels. The volume ratio of microvessels, including sprouts and cords, to the nests was 0.6282:1. Group II nests: The center of these nests underwent necrosis. The peripheral cells were rather small, with no mitosis. The PCNA index was rather low; these nests grew very slowly. There were only a few surrounding microvessels with no endothelial sprouts or cords. The volume ratio of vessels to nest was 0.0657:1. CONCLUSION: Two types of cervical carcinoma nests show a close relationship and mutual effect of the growth of carcinoma nest and angiogenesis. Group I nests grow and develop well, with abundant microvessels. Thus, many tumor cells may be angiogenic and induce angiogenesis; growth of the nests seemed dependent on adequate neovascularization. Group II nests grow slowly, with a few microvessels; the center of the nests undergoes necrosis. The inadequate blood supply must be one of the important causes of necrosis. Considering that there must have been abundant neovascularization during their growth in the past, most of the microvessels must have been obliterated and then reabsorbed to make the remaining vessels so few.     

Impaired in vivo vasculogenic potential of endothelial progenitor cells in comparison to human umbilical vein endothelial cells in a spheroid-based implantation model

Objectives:  Neovascularization represents a major challenge in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of implanted cells. An attractive therapeutic approach to overcome this is based on co-implantation of endothelial cells to create vascular networks. We have investigated the potential of human endothelial progenitor cells (EPC) to form functional blood vessels in vivo in direct comparison to vascular-derived endothelial cells, represented by human umbilical vein endothelial cells (HUVEC).
Materials and methods:  EPCs were isolated from human peripheral blood, expanded in vitro and analysed in vitro for phenotypical and functional parameters. In vivo vasculogenic potential of EPCs and HUVECs was evaluated in a xenograft model where spheroidal endothelial aggregates were implanted subcutaneously into immunodeficient mice.
Results:  EPCs were indistinguishable from HUVECs in terms of expression of classical endothelial markers CD31, von Willebrand factor, VE-cadherin and vascular endothelial growth factor-R2, and in their ability to endocytose acetylated low-density lipoprotein. Moreover, EPCs and HUVECs displayed almost identical angiogenic potential in vitro , as assessed by in vitro Matrigel sprouting assay. However in vivo , a striking and unexpected difference between EPCs and HUVECs was detected. Whereas implanted HUVEC spheroids gave rise to formation of a stable network of perfused microvessels, implanted EPC spheroids showed significantly impaired ability to form vascular structures under identical experimental conditions.
Conclusion:  Our results indicate that vascular-derived endothelial cells, such as HUVECs are superior to EPCs in terms of promoting in vivo vascularization of engineered tissues.     

Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells

In embryogenesis, coronary blood vessels are formed by vasculogenesis from epicardium-derived progenitors. Subsequently, growing or regenerating myocardium increases its vasculature by angiogenesis, forming new vessels from the pre-existing ones. Recently, cell therapies for myocardium ischemia that used different protocols have given promising results, using either extra-cardiac blood vessel cell progenitors or stimulating the cardiac angiogenesis. We have questioned whether cardiomyocytes could sustain both vasculogenesis and angiogenesis. We used a 3D culture model of tissue-like spheroids in co-cultures of cardiomyocytes supplemented either with endothelial cells or with bone marrow-derived mesenchymal stroma cells. Murine foetal cardiomyocytes introduced into non-adherent U-wells formed 3D contractile structures. They were coupled by gap junctions. Cardiomyocytes segregated inside the 3D structure into clumps separated by connective tissue septa, rich in fibronectin. Three vascular endothelial growth factor isoforms were produced (VEGF 120, 164 and 188). When co-cultured with human umbilical cord endothelial cells, vascular structures were produced in fibronectin-rich external layer and in radial septa, followed by angiogenic sprouting into the cardiomyocyte microtissue. Presence of vascular structures led to the maintenance of long-term survival and contractile capacity of cardiac microtissues. Conversely, bone marrow mesenchymal cells formed isolated cell aggregates, which progressively expressed the endothelial markers von Willebrand's antigen and CD31. They proceeded to typical vasculogenesis forming new blood vessels organised in radial pattern. Our results indicate that the in vitro 3D model of cardiomyocyte spheroids provides the two basic elements for formation of new blood vessels: fibronectin and VEGF. Within the myocardial environment, endothelial and mesenchymal cells can proceed to formation of new blood vessels either through angiogenesis or vasculogenesis, respectively.     

Ultrastructural localization of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptor-2 (FLK-1, KDR) in normal mouse kidney and in the hyperpermeable vessels induced by VPF/VEGF-expressing tumors and adenoviral vectors.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) interacts with two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, to increase microvascular permeability and induce angiogenesis. Both receptors are selectively expressed by vascular endothelial cells and are strikingly increased in tumor vessels. We used a specific antibody to localize VEGFR-2 (FLK-1, KDR) in microvascular endothelium of normal mouse kidneys and in the microvessels induced by the TA3/St mammary tumor or by infection with an adenoviral vector engineered to express VPF/VEGF. A pre-embedding method was employed at the light and electron microscopic levels using either nanogold or peroxidase as reporters. Equivalent staining was observed on both the luminal and abluminal surfaces of tumor- and adenovirus-induced vascular endothelium, but plasma membranes at interendothelial junctions were spared except at sites connected to vesiculovacuolar organelles (VVOs). VEGFR-2 was also localized to the membranes and stomatal diaphragms of some VVOs. This staining distribution is consistent with a model in which VPF/VEGF increases microvascular permeability by opening VVOs to allow the transendothelial cell passage of plasma and plasma proteins.     

Spontaneous in vitro angiogenesis in a trout pronephric stromal cell line (TPS), and in TPS-haemopoietic co-cultures

This study describes angiogenic processes taking place in the in vitro micro-environment of a trout pronephric stroma cell line (TPS) under specific culture conditions, in which fetal calf serum, horse serum and hydrocortisone-sodium-21-hemisuccinate were used as supplements to the culture medium. When TPS cultures were kept in the same flask, i.e. without passages, for longer than 7 months, epithelioid cells differentiated into endothelial cells. Early stages of such differentiation were characterised by the presence of intracellular tubular vacuoles in clusters of neighbouring epithelioid cells. Subsequently, the endothelial cells reorganised and gave rise to microvascular structures, which branched over and into the TPS multilayers. The lining cells of the microvasculature showed typical characteristics of endothelial cells, such as ovoid or cubical shape, bundles of microfilaments and microtubules, and particularly numerous small vesicles at the apical pole, some of them fused to the plasma membrane. Similar angiogenic processes were also observed in long-term haemopoietic co-cultures formed by the TPS cell line and trout pronephric cell suspensions. Developing haemopoietic cells were observed at the basal pole of the vessels, and in the vascular lumen, where some immature cells appeared in close contact with the endothelium. These results indicate that the TPS cell line contains endothelial cell precursors, which are able to differentiate under certain culture conditions.     

Human cytotrophoblasts promote endothelial survival and vascular remodeling through secretion of Ang2, PlGF, and VEGF-C

Cytotrophoblasts are specialized epithelial cells of the human placenta that differentiate to acquire tumor-like properties that allow them to invade the uterus. Concurrently, they develop endothelial-like characteristics. This transformation allows cytotrophoblasts to replace the maternal cells that line uterine vessels, thereby diverting maternal blood to the placenta. Previously, we showed that invading cytotrophoblasts secrete VEGF-C and PlGF, factors that regulate their acquisition of an endothelial-like phenotype. Here, we examined the cells' expression of angiopoietin ligands and their Tie receptors. The data show that cytotrophoblasts predominantly expressed Ang2. We also studied the paracrine functions of Ang2 and the VEGFs by culturing uterine microvascular endothelial cells in cytotrophoblast-conditioned medium, which supported their growth. Removal of VEGF-C, PlGF, and/or Ang2 from the medium caused a marked reduction in cell number due to massive apoptosis. We also assayed the angiogenic potential of cytotrophoblast-derived factors in the chick chorioallantoic membrane assay. The results showed that they stimulated angiogenesis to a level comparable to that of basic FGF. These data suggest that invasive human cytotrophoblasts use an unusual repertoire of factors to influence the angiogenic state of maternal blood vessels and that this cross talk plays an important part in the endovascular component of uterine invasion.     

Role of haematopoietic cells and endothelial progenitors in tumour angiogenesis

Bone marrow-derived cells include haematopoietic cell lineages and the recently described endothelial progenitor cells (EPCs). It has been recently emphasised that these marrow-derived cells contribute to tumour angiogenesis, and different mechanisms have been proposed that account for this activity. Whereas haematopoietic cells may promote tumour angiogenesis through the release of proangiogenic factors or by creating permissive conditions in the tumour microenvironment that favour the growth of locally derived blood vessels ("paracrine" role), endothelial progenitors are thought to directly incorporate into nascent blood vessels as bona fide endothelial cells ("building block" role). The relative contribution of these distinct pathways to tumour angiogenesis is the subject of intense investigation and debate.     

Tumor autocrine motility factor is an angiogenic factor hat stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine that primarily stimulates tumor cell motility via receptor-mediated signaling pathways and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for anti-angiogenic treatment.