Endothelial Tube Network Research Articles

Hyperactive mTORC1 in lung mesenchyme induces endothelial cell dysfunction and pulmonary vascular remodeling.

Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations in pulmonary mesenchymal cells, resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of patients with LAM develop pulmonary vascular remodeling and pulmonary hypertension. Little, however, is known regarding how LAM cells communicate with endothelial cells (ECs) to trigger vascular remodeling. In end-stage LAM lung explants, we identified EC dysfunction characterized by increased EC proliferation and migration, defective angiogenesis, and dysmorphic endothelial tube network formation. To model LAM disease, we used an mTORC1 gain-of-function mouse model with a Tsc2 KO (Tsc2KO) specific to lung mesenchyme (Tbx4LME-Cre Tsc2fl/fl), similar to the mesenchyme-specific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from mice exhibited marked transcriptomic changes despite an absence of morphological changes to the distal lung microvasculature. In contrast, 1-year-old Tbx4LME-Cre Tsc2fl/fl mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. Single-cell RNA-Seq of 1-year-old mouse lung cells identified paracrine ligands originating from Tsc2KO mesenchyme, which can signal through receptors in arterial ECs. These ECs had transcriptionally altered genes including those in pathways associated with blood vessel remodeling. The proposed pathophysiologic mesenchymal ligand-EC receptor crosstalk highlights the importance of an altered mesenchymal cell/EC axis in LAM and other hyperactive mTORC1-driven diseases. Since ECs in patients with LAM and in Tbx4LME-Cre Tsc2fl/fl mice did not harbor TSC2 mutations, our study demonstrates that constitutively active mTORC1 lung mesenchymal cells orchestrated dysfunctional EC responses that contributed to pulmonary vascular remodeling.

On-Site Construction of a Full-Thickness Skin Equivalent with Endothelial Tube Networks via Multilayer Electrospinning for Wound Coverage.

Novel full-thickness skin substitutes are of increasing interest due to the inherent limitations of current models lacking capillary networks. Herein, we developed a novel full-thickness skin tissue containing blood capillary networks through a layer-by-layer assembly approach using a handy electrospinning apparatus and evaluated its skin wound coverage potential in vivo. The average diameter and thickness of fabricated poly-ε-caprolactone-cellulose acetate scaffolds were easily tuned in the range of 474 ± 77-758 ± 113 nm and 9.43 ± 2.23-29.96 ± 5.78 μm by varying electrospinning distance and duration, as indicated by FE-SEM. Besides, keratinocytes exhibited homogeneous differentiation throughout the fibrous matrix prepared with electrospinning distance and duration of 9 cm and 1.5 min within five-layer (5L) epidermal tissues with thickness of 135-150 μm. Moreover, coculture of vascular endothelial cells, circulating fibrocytes, and fibroblasts within the 5L dermis displayed network formation in vitro, resulting in reduced inflammatory factor levels and enhanced integration with the host vasculature in vivo. Additionally, the skin equivalent grafts consisting of the epidermal layer, biomimetic basement membrane, and vascularized dermis layer with an elastic modulus of approximately 11.82 MPa exhibited accelerated wound closure effect indicative of re-epithelialization and neovascularization with long-term cell survival into the host, which was confirmed by wound-healing rate, bioluminescence imaging activity, and histological analysis. It is the first report of a full-thickness skin equivalent constructed using a battery-operated electrospinning apparatus, highlighting its tremendous potential in regenerative medicine.

Tropoelastin Promotes the Formation of Dense, Interconnected Endothelial Networks.

Tropoelastin, the soluble precursor of elastin, has been used for regenerative and wound healing purposes and noted for its ability to accelerate wound repair by enhancing vascularization at the site of implantation. However, it is not clear whether these effects are directly due to the interaction of tropoelastin with endothelial cells or communicated to endothelial cells following interactions between tropoelastin and neighboring cells, such as mesenchymal stem cells (MSCs). We adapted an endothelial tube formation assay to model in vivo vascularization with the goal of exploring the stimulatory mechanism of tropoelastin. In the presence of tropoelastin, endothelial cells formed less tubes, with reduced spreading into capillary-like networks. In contrast, conditioned media from MSCs that had been cultured on tropoelastin enhanced the formation of more dense, complex, and interconnected endothelial tube networks. This pro-angiogenic effect of tropoelastin is mediated indirectly through the action of tropoelastin on co-cultured cells. We conclude that tropoelastin inhibits endothelial tube formation, and that this effect is reversed by pro-angiogenic crosstalk from tropoelastin-treated MSCs. Furthermore, we find that the known in vivo pro-angiogenic effects of tropoelastin can be modeled in vitro, highlighting the value of tropoelastin as an indirect mediator of angiogenesis.

Fabrication of Spheroids with Dome-Shaped Endothelial Tube Networks by an Adhesive Culture System.

3D functional tissues, such as spheroids fabricated by mesenchymal stem cells (MSCs), which can mimic parts of tissues and organs, have recently been extensively studied in the fields of regenerative medicine and drug discovery. In this study, spheroids containing endothelial tubular structures are fabricated by use of a novel 3D culture plate, "MicoCell." As MicoCell has a mild cell adhesive surface and multicavity structures, it can provide multiple attached spheroids at the same time (about ≈102 to ≈104 spheroids). Spheroids can be fabricated without using serum, and are easily collected by simple pipetting and no use of enzyme. For the fabrication of spheroids containing endothelial tubular structures, MSCs and endothelial cells are co-cultured with MicoCell. Surprisingly, endothelial tubular structures are found to extend upward from the bottom where the spheroids attach onto, forming a dome-shaped morphology. Notably, some tubular structures in the spheroids have a basement membrane and markedly improved oxygen level of the inner part of spheroids. Moreover, as spheroids attach onto the bottom, they do not require any pre-treatment such as embedding into gel before microscopic observation using an optical clearing reagent. These results indicate that the culture plates will be suitable for clinical and pharmaceutical applications.

β2-Adrenergic receptor expression in human endometrial endothelial cells potentially mediates 2-hydroxyestradiol-induced microvascular stabilization: implications for endometriosis

Catecholestrogens are biologically active metabolites of 17β-estradiol (E2) synthesized by cytochrome P450 enzymes forming 2-Hydroxyestradiol (OHE2) and 4-OHE2. Although catecholestrogens bind estrogen receptors (ERs) with low affinity, their high affinity for β2-adrenergic receptors (β2-AR) regulates uterine endothelial cell proliferation in gestation1. Women with endometriosis have aberrant E2 metabolizing enzymes potentially inducing catecholestrogen production2. We hypothesize that ectopic and eutopic endometrial microvascular endothelial cells express β2-AR and that, under stimulation by 2-OHE2, endometrial endothelial cells have increased cell viability and an effect on endothelial tube stabilization. Paired eutopic/ectopic endometrial sections from women with endometriosis in the proliferative (n=5) or secretory (n=4) phases were immunostained for β2-AR and evaluated semi-quantitatively by HSCORE. Confluent human endometrial endothelial cells (HEEC) grown in 96-well plates were pretreated with control or with β-AR antagonist propranolol (2x10-6 M) followed by treatment with vehicle (control) or with 10-8 M E2 or 2-OHE2. Then, vascular tube formation (15x103 cells/well, n=3) and endothelial proliferation (5x103 cells/well, n=3) were evaluated. Tube formation was analyzed at 2, 4, 8, 16, and 24h post-treatment. Images were utilized to score endothelial tube formation and degradation based on the following scores: initial tube coalescence= 1, initiation of tube networks= 2, completed tube networks= 3, vascular destabilization= 4, and complete loss of vascular network= 5. XTT assays measured cell viability as an indicator of proliferation at 24h post-treatment. Statistical analysis used a t-test or One-way ANOVA and post hoc Tukey test. Immunohistochemistry analysis revealed increased β2-AR staining in vascular endothelial cells in ectopic compared to eutopic tissue during the proliferative phase (P<0.05) and showed a trend toward increased β2-AR staining in eutopic compared to ectopic tissue in the secretory phase; however, it did not achieve significance (P=0.055). In vitro endothelial tube formation analyses revealed no difference in scores at 2, 4, and 8h post-treatment with both E2 and 2-OHE2 compared with control. However, a difference at 16 and 24h post-treatment existed in that treatment with E2 and 2-OHE2 led to lower scores (mean = 3) compared to control (mean = 5) indicating a stabilization of endothelial tube structure in the treatment groups. XTT analyses revealed that compared to control, there were no significant differences in proliferative index in the treatment groups compared to control (P>0.05).Figure 2Endothelial Tube Formation Assay showing control compared to E2 10-8 M or 2-OHE2 10-8 M at hours 2, 4, 8, 16, and 24h post-treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The increased β2-AR expression in endothelial cells in ectopic endometriotic tissue during the proliferative phase suggests it plays a role in angiogenesis during implantation of ectopic endometrial tissue. Our in vitro data, that stabilization of newly formed endothelial tube network by 2-OHE2, suggest that the effect of 2-OHE2 may be mediated by β2-AR on endometriotic endothelial cells to maturate newly formed vasculatures during angiogenesis, thereby contributing to endometriosis pathogenesis.

In vitro and in vivo characterization of the actin polymerizing compound chondramide as an angiogenic inhibitor.

Inhibiting angiogenesis is a major approach in tumour therapy. To combat angiogenesis, the tubulin cytoskeleton has emerged as an interesting target in many pre- and clinical studies. Contrarily, the actin cytoskeleton has been largely neglected as a potential drug target in angiogenesis. However, due to the development of drug resistances, new therapeutic strategies are always needed in tumour treatment. Therefore, the therapeutic potential of actin-binding small molecules is of particular interest. We investigate the impact of chondramide (Ch), an actin polymerizing myxobacterial compound, on angiogenesis and underlying signalling. Chondramide treatment not only reduces the migration of endothelial cells but also the maturation of endothelial tube networks on matrigel. These observations can partly be explained by a disintegration of stress fibres due to aggregation and subsequent accumulation of actin in cellular structures known as 'aggresomes'. Chondramide treatment impairs the maturation of focal adhesions and reduces the amount of active β1 integrin at the cell surface. Accordingly, signalling events downstream of focal adhesions are reduced. Thus, we observed that the activity of Src and downstream factors Rho-GTPases Rac1 and Rho is reduced upon Ch treatment. In vivo, Ch was well tolerated in mice and vascularization of a tumour xenograft as well as of the developing retina was significantly reduced. Chondramide diminishes angiogenesis via two ways: (i) the disintegration of stress fibres and (ii) the reduction of promigratory signals. Our findings highlight Ch as a novel class of therapeutic lead compound with anti-angiogenic potential.

Oriented matrix promotes directional tubulogenesis

Detailed control over the structural organization of scaffolds and engineered tissue constructs is a critical need in the quest to engineer functional tissues using biomaterials. This work presents a new approach to spatially direct endothelial tubulogenesis. Micropatterned fibronectin substrates were used to control lung fibroblast adhesion and growth and the subsequent deposition of fibroblast-derived matrix during culture. The fibroblast-derived matrix produced on the micropatterned substrates was tightly oriented by these patterns, with an average variation of only 8.5°. Further, regions of this oriented extracellular matrix provided directional control of developing endothelial tubes to within 10° of the original micropatterned substrate design. Endothelial cells seeded directly onto the micropatterned substrate did not form tubes. A metric for matrix anisotropy showed a relationship between the fibroblast-derived matrix and the endothelial tubes that were subsequently developed on the same micropatterns with a resulting aspect ratio over 1.5 for endothelial tubulogenesis. Micropatterns in “L” and “Y” shapes were used to direct endothelial tubes to turn and branch with the same level of precision. These data demonstrate that anisotropic fibroblast-derived matrices instruct the alignment and shape of endothelial tube networks, thereby introducing an approach that could be adapted for future design of microvascular implants featuring organ-specific natural matrix that patterns microvascular growth.

Coordinating cell behaviour during blood vessel formation

The correct development of blood vessels is crucial for all aspects of tissue growth and physiology in vertebrates. The formation of an elaborate hierarchically branched network of endothelial tubes, through either angiogenesis or vasculogenesis, relies on a series of coordinated morphogenic events, but how individual endothelial cells adopt specific phenotypes and how they coordinate their behaviour during vascular patterning is unclear. Recent progress in our understanding of blood vessel formation has been driven by advanced imaging techniques and detailed analyses that have used a combination of powerful in vitro, in vivo and in silico model systems. Here, we summarise these models and discuss their advantages and disadvantages. We then review the different stages of blood vessel development, highlighting the cellular mechanisms and molecular players involved at each step and focusing on cell specification and coordination within the network.

Rapid Construction of Three‐Dimensional Multilayered Tissues with Endothelial Tube Networks by the Cell‐Accumulation Technique

Rapid Construction of Three‐Dimensional Multilayered Tissues with Endothelial Tube Networks by the Cell‐Accumulation Technique

Lymphatic reprogramming of microvascular endothelial cells by CEA-related cell adhesion molecule-1 via interaction with VEGFR-3 and Prox1

Here, we demonstrate that carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed and co-localized with podoplanin in lymphatic endothelial cells (LECs) of tumor but not of normal tissue. CEACAM1 overexpression in human dermal microvascular endothelial cells (HDMECs) results in a significant increase of podoplanin-positive cells in fluorescence-activated cell sorting analyses, while such effects are not observed in CEACAM1 overexpressing human umbilical vein endothelial cell (HUVECs). This effect of CEACAM1 is ceased when HDMECs are transfected with CEACAM1/y- missing the tyrosine residues in its cytoplasmic domain. CEACAM1 overexpression in HDMECs leads to an up-regulation of vascular endothelial growth factor C, -D (VEGF-C, -D) and their receptor vascular endothelial growth factor receptor 3 (VEGFR-3) at mRNA and protein levels. HDMECs transfected with CEACAM1 but not those with CEACAM1/y- show enhanced expression of the lymphatic markers Prox1, podoplanin, and LYVE-1. Furthermore, Prox1 silencing in HDMECs via small interfering RNA blocks the CEACAM1-induced increase of VEGFR-3 expression. Number and network of endothelial tubes induced by VEGF-C and -D are enhanced in CEACAM1-overexpressing HDMECs. Moreover, VEGF-A treatment of CEACAM1-silenced HDMECs restores their survival but not that with VEGF-C and VEGF-D. These data imply that the interaction of CEACAM1 with Prox1 and VEGFR-3 plays a crucial role in tumor lymphangiogenesis and reprogramming of vascular endothelial cells to LECs. CEACAM1-induced signaling effects appear to be dependent on the presence of tyrosine residues in the CEACAM1 cytoplasmic domain.

Mechanisms of Integrin–Vascular Endothelial Growth Factor Receptor Cross-Activation in Angiogenesis

The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

The release of nitric oxide from S-nitrosothiols promotes angiogenesis.

BackgroundFree nitric oxide (NO) reacts with sulphydryl residues to form S-nitrosothiols, which act as NO reservoirs. We sought to determine whether thiol-preserving agents and antioxidants, such as dithiothreitol (DTT) and vitamin C, induce NO release from S-nitrosylated proteins in endothelial cell cultures to promote angiogenesis.Methodology/Principal FindingsNO release was measured directly in cell supernatants using a Sievers NO Analyser, and in vitro angiogenesis was assessed by quantifying capillary-like tube network formation of porcine aortic endothelial cells (PAEC) on growth factor-reduced Matrigel. Incubation of PAEC with DTT or vitamin C significantly increased NO release in a concentration-dependent manner. However, the nitric oxide synthase (NOS) inhibitors, L-NNA and L-NIO, had no effect on DTT- or vitamin C-induced NO release, and there was no concomitant increase in the phosphorylation of endothelial NOS at serine-1177 following DTT or vitamin C treatment. DTT and vitamin C increased capillary-like tube network formation by nine- and two-fold, respectively, and the addition of copper ions doubled the effect of vitamin C. Surprisingly, DTT maintained endothelial tube networks for up to one month under serum-free conditions, and selective inhibitors of guanylyl cyclase (ODQ) and PKG (KT-5823) blocked this, demonstrating the requirement of cyclic GMP and PKG in this process.Conclusions/SignificanceBoth DTT and vitamin C are capable of releasing sufficient NO from S-nitrosothiols to induce capillary morphogenesis. This study provides the first evidence that increased denitrosylation leads to increased bioavailability of NO, independent of NOS activity, to promote sustained angiogenesis.

Hedgehog signaling is essential for endothelial tube formation during vasculogenesis.

During embryonic development, the first blood vessels are formed through the aggregation and subsequent assembly of angioblasts (endothelial precursors) into a network of endothelial tubes, a process known as vasculogenesis. These first vessels generally form in mesoderm that is adjacent to endodermal tissue. Although specification of the angioblast lineage is independent of endoderm interactions, a signal from the endoderm is necessary for angioblasts to assemble into a vascular network and to undergo vascular tube formation. In this study, we show that endodermally derived sonic hedgehog is both necessary and sufficient for vascular tube formation in avian embryos. We also show that Hedgehog signaling is required for vascular tube formation in mouse embryos, and for vascular cord formation in cultured mouse endothelial cells. These results demonstrate a previously uncharacterized role for Hedgehog signaling in vascular development, and identify Hedgehog signaling as an important component of the molecular pathway leading to vascular tube formation.

Vascular development and disorders: Molecular analysis and pathogenic insights

Blood vessels are among the first organs to develop during embryogenesis and are essential for organogenesis and nutrition of the embryo. Although formation of blood vessels most actively occurs during embryonic development, tissue vascularization proceeds after birth in the retina, in the heart, and, cyclically, in the reproductive organs. In addition, abnormal vessel growth importantly contributes to the pathogenesis of several disorders with high morbidity and mortalitiy. Excessive vessel growth has been implicated in the pathogenesis of retinopathies, cancer and inflammation. In contrast, insufficient vessel growth may lead to tissue ischemia and failure. Although formation of new blood vessels has typically been associated with endothelial cells, the periendothelial mural pericytes (smaller vessels) or smooth muscle cells (larger vessels) are equally important for normal and pathological vessel formation. Indeed, a developing network of endothelial tubes that is not surrounded by mural cells is fragile and prone to rupture, remains susceptible to hypoxic regulation, fails to become remodeled, is unable to sustain proper circulation, and cannot adapt to changes in physiological demands of blood supply. Notably, vascular growth does not always involve formation of new capillaries, but may depend on remodeling of pre-existing arteriolized vessels, such as during collateral growth in the myocardium. The importance of periendothelial cells is further deduced from the pathological loss of smooth muscle cells in atherosclerotic vessels (leading to media necrosis), or from the pericyte drop out in diabetic retinal vessels, which renders them prone to aneurysmal rupture. Conversely, accumulation of smooth muscle cells contributes to atherosclerotic plaque growth and arterial stenosis. Recent studies have highlighted the importance of endothelial periendothelial cell crosstalk during normal and pathological blood vessel formation. Accordingly, integrated research into the molecular mechanisms that regulate the development and function of both endothelial and pericyte/smooth muscle cells has become a major focus in vascular biology. A number of candidate molecules (growth factors, matrix component, adhesion molecules, and proteinases) have been identified that stimulate or inhibit these processes. Recently, remarkable progress in their molecular analysis has been achieved through targeted manipulation of the mouse genome. Their role will be discussed in this review.