Quiescent Vasculature Research Articles

Mechanosensitive mTORC1 signaling maintains lymphatic valves.

Homeostatic maintenance and repair of lymphatic vessels are essential for health. We investigated the dynamics and the molecular mechanisms of lymphatic endothelial cell (LEC) renewal in adult mesenteric quiescent lymphatic vasculature using label-retention, lineage tracing, and cell ablation strategies. Unlike during development, adult LEC turnover and proliferation was confined to the valve regions of collecting vessels, with valve cells displaying the shortest lifespan. Proliferating valve sinus LECs were the main source for maintenance and repair of lymphatic valves. We identified mechanistic target of rapamycin complex 1 (mTORC1) as a mechanoresponsive pathway activated by fluid shear stress in LECs. Depending on the shear stress level, mTORC1 activity drives division of valve cells or dictates their mechanic resilience through increased protein synthesis. Overactivation of lymphatic mTORC1 in vivo promoted supernumerary valve formation. Our work provides insights into the molecular mechanisms of maintenance of healthy lymphatic vascular system.

Notch Signaling in the Vasculature: Angiogenesis and Angiocrine Functions.

Formation of a functional blood vessel network is a complex process tightly controlled by pro- and antiangiogenic signals released within the local microenvironment or delivered through the bloodstream. Endothelial cells precisely integrate such temporal and spatial changes in extracellular signals and generate an orchestrated response by modulating signaling transduction, gene expression, and metabolism. A key regulator in vessel formation is Notch signaling, which controls endothelial cell specification, proliferation, migration, adhesion, and arteriovenous differentiation. This review summarizes the molecular biology of endothelial Notch signaling and how it controls angiogenesis and maintenance of the established, quiescent vasculature. In addition, recent progress in the understanding of Notch signaling in endothelial cells for controlling organ homeostasis by transcriptional regulation of angiocrine factors and its relevance to disease will be discussed.

Strategies to Attenuate Myocardial Infarction and No-Reflow Through Preservation of Vascular Integrity by Pigment Epithelium-Derived Factor.

The phenomenon of no-reflow seriously limits the therapeutic value of coronary recanalization and leads to poor prognosis. Recent studies have demonstrated the potential role of pigment epithelium-derived factor (PEDF) in stabilizing endothelial cell junction, reducing vascular permeability and maintaining a quiescent vasculature. In this study, intramyocardial gene delivery was performed 5 days before the acute myocardial infarction/recanalization experiment in male rats. Positron emission tomography perfusion imaging with 13N-NH3 indicated PEDF to promote microvascular reperfusion significantly 4 h postcoronary occlusion. PEDF was observed to maintain the stability of endothelial adherens junctions (AJs), thus preventing the occurrence of no-reflow. PEDF reduced the hypoxia-induced vascular endothelial (VE)-cadherin endocytosis through PEDF/LR/Src/VE-cadherin S665 axis in vitro, which was remarkably observed to maintain endothelial AJs. Generally, PEDF might function as a relevant target for therapeutic vasculoprotection by way of regulating the phosphorylation level of VE-cadherin according to our data, thus being crucial for preventing no-reflow.

Largazole Inhibits Ocular Angiogenesis by Modulating the Expression of VEGFR2 and p21.

Ocular angiogenic diseases, characterized by abnormal blood vessel formation in the eye, are the leading cause of blindness. Although Anti-VEGF therapy is the first-line treatment in the market, a substantial number of patients are refractory to it or may develop resistance over time. As uncontrolled proliferation of vascular endothelial cells is one of the characteristic features of pathological neovascularization, we aimed to investigate the role of the class I histone deacetylase (HDAC) inhibitor Largazole, a cyclodepsipeptide from a marine cyanobacterium, in ocular angiogenesis. Our study showed that Largazole strongly inhibits retinal vascular endothelial cell viability, proliferation, and the ability to form tube-like structures. Largazole strongly inhibits the vessel outgrowth from choroidal explants in choroid sprouting assay while it does not affect the quiescent choroidal vasculature. Largazole also inhibits vessel outgrowth from metatarsal bones in metatarsal sprouting assay without affecting pericytes coverage. We further demonstrated a cooperative effect between Largazole and an approved anti-VEGF drug, Alflibercept. Mechanistically, Largazole strongly inhibits the expression of VEGFR2 and leads to an increased expression of cell cycle inhibitor, p21. Taken together, our study provides compelling evidence on the anti-angiogenic role of Largazole that exerts its function through mediating different signaling pathways.

Reporter mice for isolating and auditing cell type-specific extracellular vesicles in vivo.

Extracellular vesicles (EVs) are abundant, lipid-enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type-specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR-Cas9-mediated genome editing to generate mice bearing a CD63-emGFPloxP/stop/loxP knock-in cassette that enables the specific labeling of circulating CD63+ vesicles from any cell type when crossed with lineage-specific Cre recombinase driver mice. As proof-of-principle, we have crossed these mice with Cdh5-CreERT2 mice to generate CD63emGFP+ vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma-purified EVs contain a subpopulation of emGFP+ vesicles that are derived from the endothelium, co-express additional EV (e.g., CD9 and CD81) and endothelial cell (e.g., CD105) markers, and they harbor specific miRNAs (e.g., miR-126, miR-30c, and miR-125b). This new mouse strain should be a useful genetic tool for generating cell type-specific, CD63+ EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.

Bioengineering of Pulmonary Epithelium With Preservation of the Vascular Niche.

The shortage of transplantable donor organs directly affects patients with end-stage lung disease, for which transplantation remains the only definitive treatment. With the current acceptance rate of donor lungs of only 20%, rescuing even one half of the rejected donor lungs would increase the number of transplantable lungs threefold, to 60%. We review recent advances in lung bioengineering that have potential to repair the epithelial and vascular compartments of the lung. Our focus is on the long-term support and recovery of the lung ex vivo, and the replacement of defective epithelium with healthy therapeutic cells. To this end, we first review the roles of the lung epithelium and vasculature, with focus on the alveolar-capillary membrane, and then discuss the available and emerging technologies for ex vivo bioengineering of the lung by decellularization and recellularization. While there have been many meritorious advances in these technologies for recovering marginal quality lungs to the levels needed to meet the standards for transplantation – many challenges remain, motivating further studies of the extended ex vivo support and interventions in the lung. We propose that the repair of injured epithelium with preservation of quiescent vasculature will be critical for the immediate blood supply to the lung and the lung survival and function following transplantation.

The metabolic engine of endothelial cells.

Endothelial cells (ECs) line the quiescent vasculature but can form new blood vessels (a process termed angiogenesis) in disease. Strategies targeting angiogenic growth factors have been clinically developed for the treatment of malignant and ocular diseases. Studies over the past decade have documented that several pathways of central carbon metabolism are necessary for EC homeostasis and growth, and that strategies that stimulate or block EC metabolism can be used to promote or inhibit vessel growth, respectively. In this Review, we provide an updated overview of the growing understanding of central carbon metabolic pathways in ECs and the therapeutic opportunities for targeting EC metabolism.

Application of a Novel Murine Ear Vein Model to Evaluate the Effects of a Vascular Radioprotectant on Radiation-Induced Vascular Permeability and Leukocyte Adhesion

Vascular injury after radiation exposure contributes to multiple types of tissue injury through a cascade of events. Some of the earliest consequences of radiation damage include increased vascular permeability and promotion of inflammation, which is partially manifested by increased leukocyte-endothelial (L/E) interactions. We describe herein a novel intravital imaging method to evaluate L/E interactions, as a function of shear stress, and vascular permeability at multiple time points after local irradiation to the ear. This model permitted analysis of quiescent vasculature that was not perturbed by any surgical manipulation prior to imaging. To evaluate the effects of radiation on vascular integrity, fluorescent dextran was injected intravenously and its extravasation in the extravascular space surrounding the ear vasculature was measured at days 3 and 7 after 6 Gy irradiation. The vascular permeability rate increased approximately twofold at both days 3 and 7 postirradiation ( P < 0.05). Leukocyte rolling, which is indicative of L/E interactions, was significantly increased in mice at 24 h postirradiation compared to that of nonirradiated mice. To assess our model, as a means for assessing vascular radioprotectants, we treated additional cohorts of mice with a thrombopoietin mimetic, TPOm (RWJ-800088). In addition to stimulating platelet formation, thrombopoietin can protect vasculature after several forms of injury. Thus, we hypothesized that TPOm would reduce vascular permeability and L/E adhesion after localized irradiation to the ear vasculature of mice. If TPOm reduced these consequences of radiation, it would validate the utility of our intravital imaging method. TPOm reduced radiation-induced vascular leakage to control levels at day 7. Furthermore, L/E cell interactions were also reduced in irradiated mice treated with TPOm, compared with mice receiving irradiation alone, particularly at high shear stress ( P = 0.03, Kruskal-Wallis). We conclude that the ear model is useful for monitoring quiescent normal tissue vascular injury after radiation exposure. Furthermore, the application of TPOm, for preventing early inflammatory response created by damage to vascular endothelium, suggests that this drug may prove useful in reducing toxicities from radiotherapy, which damage microvasculature that critically important to tissue function.

Transient loss of venous integrity during developmental vascular remodeling leads to red blood cell extravasation and clearance by lymphatic vessels.

ABSTRACTMaintenance of blood vessel integrity is crucial for vascular homeostasis and is mainly controlled at the level of endothelial cell (EC) junctions. Regulation of endothelial integrity has largely been investigated in the mature quiescent vasculature. Less is known about how integrity is maintained during vascular growth and remodeling involving extensive junctional reorganization. Here, we show that embryonic mesenteric blood vascular remodeling is associated with a transient loss of venous integrity and concomitant extravasation of red blood cells (RBCs), followed by their clearance by the developing lymphatic vessels. In wild-type mouse embryos, we observed activated platelets extending filopodia at sites of inter-EC gaps. In contrast, embryos lacking the activatory C-type lectin domain family 1, member b (CLEC1B) showed extravascular platelets and an excessive number of RBCs associated with and engulfed by the first lymphatic EC clusters that subsequently form lumenized blood-filled vessels connecting to the lymphatic system. These results uncover novel functions of platelets in maintaining venous integrity and lymphatic vessels in clearing extravascular RBCs during developmental remodeling of the mesenteric vasculature. They further provide insight into how vascular abnormalities characterized by blood-filled lymphatic vessels arise.

Photo-responsive tetraether lipids based vesicles for prophyrin mediated vascular targeting and direct phototherapy

Tetraether lipids (TELs) derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius are dominated by polyisoprenoid skeleton. The unique molecular stability of TELs is attributed to the presence of cyclopentane rings, methyl side groups and sugar residues that create extensive hydrogen bond network. In addition, the presence of ether linkages and the lacking of double bonds make them an epitome candidate for photodynamic therapy (PDT). A subtle blend of formulation design to trigger efficient photo responses of protoporphyrin IX (PpIX) exploiting TELs was developed. The platform has demonstrated in principle a practical potential in PDT in terms of prompt Vascular Targeting Photodynamic therapy (VTP) in-ovo chick chorioallantoic membrane (CAM) model. Short PpIX-light interval was associated with thrombosis and massive vascular occlusion in and out the irradiated area after TEL9mol% liposomes have been intravenously injected. Profoundly, TEL62mol% liposomes have proved to be the most effective liposomes that demonstrated localized suppression of angiogenesis in the irradiated area without quiescent vasculature damage. The massive thrombotic effect was no longer observed and eventually the chick has survived. After long PpIX-light interval, TEL62mol% has deliberately gained metronomic PDT at low rate of PpIX dosimetry and the radiant exposure doses in human ovarian carcinoma (SKOV-3) cells as determined by PpIXIC50. These findings could be explained by the fact that TELs impart a remarkable stability to the liposomal bilayer that makes them a potential platform for photodynamic applications.

Pleiotropic effects of herbs characterized with blood-activating and stasis-resolving functions on angiogenesis

Accumulative evidences have underpinned the nature candidates from Chinese medicine (CM), particularly CM served as blood activating and stasis resolving (BASR, Huoxue Huayu in Chinese) by targeting tumor-associated angiogenesis. However, recent experiment research on the therapeutic angiogenesis by BASR-CM attracts wide attention and discussion. This opinion review focused on the underlying link between two indications and anticipated that (1) BASR-CM might emphasize on a balanced multi-cytokines network interaction; (2) BASR-CM might address on the nature of diseases prior to differently affecting physiological and pathological angiogenesis; (3) BASR-CM might mainly act on perivascular cells, either promotes arteriogenesis by increasing arteriogenic factors in ischemic diseases, or simultaneously keep a quiescent vasculature to impede angiogenesis in tumor context.

In Vivo Assays for Assessing the Role of the Wilms' Tumor Suppressor 1 (Wt1) in Angiogenesis.

The Wilms' tumor suppressor gene (WT1) is widely expressed during neovascularization, but it is almost entirely absent in quiescent adult vasculature. However, in vessels undergoing angiogenesis, WT1 is dramatically upregulated. Studies have shown Wt1 has a role in both tumor and ischemic angiogenesis, but the mechanism of Wt1 action in angiogenic tissue remains to be elucidated. Here, we describe two methods for induction of in vivo angiogenesis (subcutaneous sponge implantation, femoral artery ligation) that can be used to assess the influence of Wt1 on new blood vessel formation. Subcutaneously implanted sponges stimulate an inflammatory and fibrotic response including cell infiltration and angiogenesis. Femoral artery ligation creates ischemia in the distal hindlimb and produces an angiogenic response to reperfuse the limb which can be quantified in vivo by laser Doppler flowmetry. In both of these models, the role of Wt1 in the angiogenic process can be assessed using histological/immunohistochemical staining, molecular analysis (qPCR) and flow cytometry. Furthermore, combined with suitable genetic modifications, these models can be used to explore the causal relationship between Wt1 expression and angiogenesis and to trace the lineage of cells expressing Wt1. This approach will help to clarify the importance of Wt1 in regulating neovascularization in the adult, and its potential as a therapeutic target.

The Orphan Receptor Tie1 Controls Angiogenesis and Vascular Remodeling by Differentially Regulating Tie2 in Tip and Stalk Cells.

SUMMARYTie1 is a mechanistically poorly characterized endothelial cell (EC)-specific orphan receptor. Yet, Tie1 deletion is embryonic lethal and Tie1 has been implicated in critical vascular pathologies, including atherosclerosis and tumor angiogenesis. Here, we show that Tie1 does not function independently but exerts context-dependent effects on the related receptor Tie2. Tie1 was identified as an EC activation marker that is expressed during angiogenesis by a subset of angiogenic tip and remodeling stalk cells and downregulated in the adult quiescent vasculature. Functionally, Tie1 expression by angiogenic EC contributes to shaping the tip cell phenotype by negatively regulating Tie2 surface presentation. In contrast, Tie1 acts in remodeling stalk cells cooperatively to sustain Tie2 signaling. Collectively, our data support an interactive model of Tie1 and Tie2 function, in which dynamically regulated Tie1 versus Tie2 expression determines the net positive or negative effect of Tie1 on Tie2 signaling.

Genetic targeting of sprouting angiogenesis using Apln-CreER.

Under pathophysiological conditions in adults, endothelial cells (ECs) sprout from pre-existing blood vessels to form new ones by a process termed angiogenesis. During embryonic development, Apelin (APLN) is robustly expressed in vascular ECs. In adult mice, however, APLN expression in the vasculature is significantly reduced. Here we show that APLN expression is reactivated in adult ECs after ischaemia insults. In models of both injury ischaemia and tumor angiogenesis, we find that Apln-CreER genetically labels sprouting but not quiescent vasculature. By leveraging this specific activity, we demonstrate that abolishment of the VEGF–VEGFR2 signalling pathway as well as ablation of sprouting ECs diminished tumour vascularization and growth without compromising vascular homeostasis in other organs. Collectively, we show that Apln-CreER distinguishes sprouting vessels from stabilized vessels in multiple pathological settings. The Apln-CreER line described here will greatly aid future mechanistic studies in both vascular developmental biology and adult vascular diseases.

A simple method to assess in vivo proliferation in lung vasculature with EdU: the case of MMC-induced PVOD in rat.

5-Ethynyl-2′-deoxyuridine (EdU) incorporation is becoming the gold standard method for in vitro and in vivo visualization of proliferating cells. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration. It can be used to easily detect DNA replication in large tissue samples or organ explants with low proliferation and turnover of cells formerly believed to be in a “terminal” state of differentiation. Here we describe a protocol for the localization and identification of proliferating cells in quiescent or injured pulmonary vasculature, in a model of pulmonary veno-occlusive disease (PVOD). PVOD is an uncommon form of pulmonary hypertension characterized by progressive obstruction of small pulmonary veins. We previously reported that mitomycin-C (MMC) therapy is associated with PVOD in human. We demonstrated that MMC can induce PVOD in rats, which currently represents the sole animal model that recapitulates human PVOD lesions. Using the EdU assay, we demonstrated that MMC-exposed lungs displayed areas of exuberant microvascular endothelial cell proliferation which mimics pulmonary capillary hemangiomatosis, one of the pathologic hallmarks of human PVOD. In vivo pulmonary cell proliferation measurement represents an interesting methodology to investigate the potential efficacy of therapies aimed at normalizing pathologic angioproliferation.

κ Opioid receptor ligands regulate angiogenesis in development and in tumours

Opioid systems mainly regulate physiological functions such as pain, emotional tone and reward circuitry in neural tissues (brain and spinal cord). These systems are also found in extraneural tissues (ganglia, gut, spleen, stomach, lung, pancreas, liver, heart, blood and blood vessels), and recent studies have elucidated their roles in various organs. The current review focuses on the roles of opioid systems in blood vessels, especially angiogenesis, during development and tumour malignancy. The balance between endogenous activators and inhibitors of angiogenesis delicately maintains a normally quiescent vasculature to sustain homeostasis. Disturbance of this balance causes pathogenic angiogenesis and, especially in tumours, several activators such as VEGF are highly expressed in the tumour microenvironment and strongly induce tumour angiogenesis, the so-called angiogenic switch. Recently, we demonstrated that κ opioid receptor agonists function as anti-angiogenic factors, which impede the angiogenic switch, in vascular development and tumour angiogenesis by inhibiting the expression of receptors for VEGF. In clinical medicine, angiogenesis inhibitors that target VEGF signalling such as bevacizumab are used as anti-cancer drugs. Although therapies that inhibit tumour angiogenesis have been highly successful for tumour therapy, most patients eventually develop resistance to this anti-angiogenic therapy. Thus, we must identify novel targets for anti-angiogenic agents to sustain inhibition of angiogenesis for tumour therapy. The regulation of responses to κ opioid receptor ligands could be useful for controlling vascular formation under physiological conditions and in cancers, and thus could offer therapeutic benefits beyond the relief of pain. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Embelin inhibits endothelial mitochondrial respiration and impairs neoangiogenesis during tumor growth and wound healing

In the normal quiescent vasculature, only 0.01% of endothelial cells (ECs) are proliferating. However, this proportion increases dramatically following the angiogenic switch during tumor growth or wound healing. Recent evidence suggests that this angiogenic switch is accompanied by a metabolic switch. Here, we show that proliferating ECs increasingly depend on mitochondrial oxidative phosphorylation (OxPhos) for their increased energy demand. Under growth conditions, ECs consume three times more oxygen than quiescent ECs and work close to their respiratory limit. The increased utilization of the proton motif force leads to a reduced mitochondrial membrane potential in proliferating ECs and sensitizes to mitochondrial uncoupling. The benzoquinone embelin is a weak mitochondrial uncoupler that prevents neoangiogenesis during tumor growth and wound healing by exhausting the low respiratory reserve of proliferating ECs without adversely affecting quiescent ECs. We demonstrate that this can be exploited therapeutically by attenuating tumor growth in syngenic and xenograft mouse models. This novel metabolic targeting approach might be clinically valuable in controlling pathological neoangiogenesis while sparing normal vasculature and complementing cytostatic drugs in cancer treatment.

NRP1 Presented in trans to the Endothelium Arrests VEGFR2 Endocytosis, Preventing Angiogenic Signaling and Tumor Initiation

Neuropilin 1 (NRP1) modulates angiogenesis by binding vascular endothelial growth factor (VEGF) and its receptor, VEGFR2. We examined the consequences when VEGFR2 and NRP1 were expressed on the same cell (cis) or on different cells (trans). In cis, VEGF induced rapid VEGFR2/NRP1 complex formation and internalization. In trans, complex formation was delayed and phosphorylation of phospholipase Cγ (PLCγ) and extracellular regulated kinase 2 (ERK2) was prolonged, whereas ERK1 phosphorylation was reduced. Trans complex formation suppressed initiation and vascularization of NRP1-expressing mouse fibrosarcoma and melanoma. Suppression in trans required high-affinity, steady-state binding of VEGF to NRP1, which was dependent on the NRP1 C-terminal domain. Compatible with a trans effect of NRP1, quiescent vasculature in the developing retina showed continuous high NRP1 expression, whereas angiogenic sprouting occurred where NRP1 levels fluctuated between adjacent endothelial cells. Therefore, through communication in trans, NRP1 can modulate VEGFR2 signaling and suppress angiogenesis.

Notch controls retinal blood vessel maturation and quiescence

Blood vessels form a hierarchically organized network of arteries, capillaries and veins, which develops through a series of growth, pruning and maturation processes. In contrast to the rapidly increasing insight into the processes controlling vascular growth and, in particular, endothelial sprouting and proliferation, the conversion of immature vessels into a fully functional, quiescent vasculature remains little understood. Here we used inducible, cell type-specific genetic approaches to show that endothelial Notch signaling is crucial for the remodeling of veins and the perivenous capillary plexus, which occurs after the completion of the initial angiogenic growth phase in the retina of adolescent mice. Mutant vessels showed ectopic proliferation and sprouting, defective recruitment of supporting mural cells, and failed to downregulate the expression of VEGF receptors. Surprisingly, by contrast Notch was dispensable in the endothelium of remodeling postnatal arteries. Taken together, our results identify key processes contributing to vessel remodeling, maturation and the acquisition of a quiescent phenotype in the final stage of developmental angiogenesis.

Endothelial GATA-6 Deficiency Promotes Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a chronic and progressive disease characterized by pulmonary vasculopathy with elevation of pulmonary artery pressure, often culminating in right ventricular failure. GATA-6, a member of the GATA family of zinc-finger transcription factors, is highly expressed in quiescent vasculature and is frequently lost during vascular injury. We hypothesized that endothelial GATA-6 may play a critical role in the molecular mechanisms underlying endothelial cell (EC) dysfunction in PAH. Here we report that GATA-6 is markedly reduced in pulmonary ECs lining both occluded and nonoccluded vessels in patients with idiopathic and systemic sclerosis-associated PAH. GATA-6 transcripts are also rapidly decreased in rodent PAH models. Endothelial GATA-6 is a direct transcriptional regulator of genes controlling vascular tone [endothelin-1, endothelin-1 receptor type A, and endothelial nitric oxide synthase (eNOS)], pro-inflammatory genes, CX3CL1 (fractalkine), 5-lipoxygenease-activating protein, and markers of vascular remodeling, including PAI-1 and RhoB. Mice with the genetic deletion of GATA-6 in ECs (Gata6-KO) spontaneously develop elevated pulmonary artery pressure and increased vessel muscularization, and these features are further exacerbated in response to hypoxia. Furthermore, innate immune cells including macrophages (CD11b(+)/F4/80(+)), granulocytes (Ly6G(+)/CD45(+)), and dendritic cells (CD11b(+)/CD11c(+)) are significantly increased in normoxic Gata6-KO mice. Together, our findings suggest a critical role of endothelial GATA-6 deficiency in development and disease progression in PAH.