Mechanisms of skin vascular maturation and maintenance captured by longitudinal imaging of live mice.

A functional network of blood vessels is essential for organ growth and homeostasis, yet how the vasculature matures and maintains homeostasis remains elusive in live mice. By longitudinally tracking the same neonatal endothelial cells (ECs) over days to weeks, we found that capillary plexus expansion is driven by vessel regression to optimize network perfusion. Neonatal ECs rearrange positions to evenly distribute throughout the developing plexus and become positionally stable in adulthood. Upon local ablation, adult ECs survive through a plasmalemmal self-repair response, while neonatal ECs are predisposed to die. Furthermore, adult ECs reactivate migration to assist vessel repair. Global ablation reveals coordinated maintenance of the adult vascular architecture that allows for eventual network recovery. Lastly, neonatal remodeling and adult maintenance of the skin vascular plexus are orchestrated by temporally restricted, neonatal VEGFR2 signaling. Our work sheds light on fundamental mechanisms that underlie both vascular maturation and adult homeostasis in vivo.

Dll4-Notch1 signaling but not VEGF-A is essential for hyperoxia induced vessel regression in retina.

It is well recognized that decreased vascular endothelial growth factor A (VEGF-A) mRNA plays an important role in retinal vessel regression induced by hyperoxia. However, this concept has been challenged by increasing new evidence. Furthermore, VEGF-A strongly enhances Dll4 expression and inhibition of Dll4-Notch signaling leads to excessive sprouting angiogenesis. Recently, it is shown that inactivation of Dll4-Notch1 signaling reduce hyperoxia induced vessel regression. It is unknown whether sprouting angiogenesis contributes to the protective effect or not and further investigations are needed. Moreover, the expression of Dll4 or Notch1 activation in the regressing plexus remains elucidated. To determine the role of VEGF-A and Dll4-Notch1 signaling in hyperoxia induced vascular regression in the retina, we used mice at postnatal day 5 (P5) - P7. Hyperoxia induced massive vascular regression in the central plexus but not in the angiogenic plexus and had no effect on sprouting angiogenesis. Immunostaining showed that VEGF-A was significantly repressed in the angiogenic front region after hyperoxia exposure but not detectable in the central area of both normoxia and hyperoxia treated retinas. In contrast, Notch ligand Delta-like 4 (Dll4) and Notch1 intracellular domain (N1-ICD) expression were inhibited in the regressing capillaries of central retina but comparable in the angiogenic plexus after high oxygen treatment. Moreover, administration of Dll4 neutralizing antibody or γ-Secretase inhibitor DAPT significantly aggravated vessel regression induced by short-time hyperoxia administration. Our data show that repressed Dll4-Notch1 signaling pathway but not downregulation of VEGF-A expression are responsible for hyperoxia induced pervasive vessel regression.

A mathematical model of angiogenesis and tumor growth: analysis and application in anti-angiogenesis therapy.

The purpose of this paper is to develop a new coupled mathematical model of angiogenesis (new blood vessel growth) and tumor growth to study cancer development and anti-angiogenesis therapy. The angiogenesis part assumes the capillary to be a viscoelastic continuum whose stress depends on cell proliferation or death, and the tumor part is a Darcy's law model regarding the tumor mass as an incompressible fluid where the nutrient-dependent growth elicits volume change. For the coupled model, we provide both an inviscid analysis and a parameter sensitivity analysis of the angiogenesis model in response to a stationary hypoxic tumor, and a steady state analysis of the tumor growth in response to a fixed and long blood capillary. The analysis shows that the stable steady state tumor with an invading blood capillary exists if and only if the nutrient release rate divided by the decay rate is less than the tumor viable limit, and the full tumor encloses one part of the capillary in this steady state. Afterwards, we use the coupled model to simulate vascularized tumor growth and anti-angiogenesis therapy. The simulations show that the tumor tends to maximize the nutrient transfer by blood vessel co-option and the anti-angiogenesis treatment by using growth factor neutralizing antibodies would regress the neovasculature and shrink the tumor size. However, the shrunken tumor mass could survive by feeding on mature blood vessels that resist the treatment. This implies the limited efficacy of the anti-angiogenesis monotherapy and its effect on vessel normalization.

Endothelial cell-derived non-canonical Wnt ligands control vascular pruning in angiogenesis.

Multiple cell types involved in the regulation of angiogenesis express Wnt ligands. Although ß-catenin dependent and independent Wnt signaling pathways have been shown to control angiogenesis, the contribution of individual cell types to activate these downstream pathways in endothelial cells (ECs) during blood vessel formation is still elusive. To investigate the role of ECs in contributing Wnt ligands for regulation of blood vessel formation, we conditionally deleted the Wnt secretion factor Evi in mouse ECs (Evi-ECKO). Evi-ECKO mice showed decreased microvessel density during physiological and pathological angiogenesis in the postnatal retina and in tumors, respectively. The reduced microvessel density resulted from increased vessel regression accompanied by decreased EC survival and proliferation. Concomitantly, survival-related genes were downregulated and cell cycle arrest- and apoptosis-inducing genes were upregulated. EVI silencing in cultured HUVECs showed similar target gene regulation, supporting a mechanism of EC-derived Wnt ligands in controlling EC function. ECs preferentially expressed non-canonical Wnt ligands and canonical target gene expression was unaffected in Evi-ECKO mice. Furthermore, the reduced vascularization of Matrigel plugs in Evi-ECKO mice could be rescued by introduction of non-canonical Wnt5a. Treatment of mouse pups with the non-canonical Wnt inhibitor TNP470 resulted in increased vessel regression accompanied by decreased EC proliferation, thus mimicking the proliferation-dependent Evi-ECKO remodeling phenotype. Taken together, this study identified EC-derived non-canonical Wnt ligands as regulators of EC survival, proliferation and subsequent vascular pruning during developmental and pathological angiogenesis.

Vasoprotective effect of PDGF-CC mediated by HMOX1 rescues retinal degeneration.

Blood vessel degeneration is critically involved in nearly all types of degenerative diseases. Therefore strategies to enhance blood vessel protection and survival are highly needed. In this study, using different animal models and cultured cells, we show that PDGF-CC is a potent vascular protective and survival factor. PDGF-CC deficiency by genetic deletion exacerbated blood vessel regression/degeneration in various animal models. Importantly, treatment with PDGF-CC protein not only increased the survival of retinal blood vessels in a model of oxygen-induced blood vessel regression but also markedly rescued retinal and blood vessel degeneration in a disease model of retinitis pigmentosa. Mechanistically, we revealed that heme oxygenase-1 (HMOX1) activity is critically required for the vascular protective/survival effect of PDGF-CC, because blockade of HMOX1 completely abolished the protective effect of PDGF-CC in vitro and in vivo. We further found that both PDGF receptors, PDGFR-ß and PDGFR-α, are required for the vasoprotective effect of PDGF-CC. Thus our data show that PDGF-CC plays a pivotal role in maintaining blood vessel survival and may be of therapeutic value in treating various types of degenerative diseases.

Anti-VEGF- and anti-VEGF receptor-induced vascular alteration in mouse healthy tissues.

Systemic therapy with anti-VEGF drugs such as bevacizumab is widely used for treatment of human patients with various solid tumors. However, systemic impacts of such drugs in host healthy vasculatures remain poorly understood. Here, we show that, in mice, systemic delivery of an anti-VEGF or an anti-VEGF receptor (VEGFR)-2 neutralizing antibody caused global vascular regression. Among all examined tissues, vasculatures in endocrine glands, intestinal villi, and uterus are the most affected in response to VEGF or VEGFR-2 blockades. Thyroid vascular fenestrations were virtually completely blocked by VEGF blockade, leading to marked accumulation of intraendothelial caveolae vesicles. VEGF blockade markedly increased thyroid endothelial cell apoptosis, and withdrawal of anti-VEGF resulted in full recovery of vascular density and architecture after 14 d. Prolonged anti-VEGF treatment resulted in a significant decrease of the circulating level of the predominant thyroid hormone free thyroxine, but not the minimal isoform of triiodothyronine, suggesting that chronic anti-VEGF treatment impairs thyroid functions. Conversely, VEGFR-1-specific blockade produced virtually no obvious phenotypes. These findings provide structural and functional bases of anti-VEGF-specific drug-induced side effects in relation to vascular changes in healthy tissues. Understanding anti-VEGF drug-induced vascular alterations in healthy tissues is crucial to minimize and even to avoid adverse effects produced by currently used anti-VEGF-specific drugs.

Pigment epithelium-derived factor as a multifunctional regulator of wound healing.

During dermal wound repair, hypoxia-driven proliferation results in dense but highly permeable, disorganized microvascular networks, similar to those in solid tumors. Concurrently, activated dermal fibroblasts generate an angiopermissive, provisional extracellular matrix (ECM). Unlike cancers, wounds naturally resolve via blood vessel regression and ECM maturation, which are essential for reestablishing tissue homeostasis. Mechanisms guiding wound resolution are poorly understood; one candidate regulator is pigment epithelium-derived factor (PEDF), a secreted glycoprotein. PEDF is a potent antiangiogenic in models of pathological angiogenesis and a promising cancer and cardiovascular disease therapeutic, but little is known about its physiological function. To examine the roles of PEDF in physiological wound repair, we used a reproducible model of excisional skin wound healing in BALB/c mice. We show that PEDF is abundant in unwounded and healing skin, is produced primarily by dermal fibroblasts, binds to resident microvascular endothelial cells, and accumulates in dermal ECM and epidermis. PEDF transcript and protein levels were low during the inflammatory and proliferative phases of healing but increased in quantity and colocalization with microvasculature during wound resolution. Local antibody inhibition of endogenous PEDF delayed vessel regression and collagen maturation during the remodeling phase. Treatment of wounds with intradermal injections of exogenous, recombinant PEDF inhibited nascent angiogenesis by repressing endothelial proliferation, promoted vascular integrity and function, and increased collagen maturity. These results demonstrate that PEDF contributes to the resolution of healing wounds by causing regression of immature blood vessels and stimulating maturation of the vascular microenvironment, thus promoting a return to tissue homeostasis after injury.

Lymphatic Vessel Regression and Its Therapeutic Applications: Learning From Principles of Blood Vessel Regression.

Aberrant lymphatic system function has been increasingly implicated in pathologies such as lymphedema, organ transplant rejection, cardiovascular disease, obesity, and neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. While some pathologies are exacerbated by lymphatic vessel regression and dysfunction, induced lymphatic regression could be therapeutically beneficial in others. Despite its importance, our understanding of lymphatic vessel regression is far behind that of blood vessel regression. Herein, we review the current understanding of blood vessel regression to identify several hallmarks of this phenomenon that can be extended to further our understanding of lymphatic vessel regression. We also summarize current research on lymphatic vessel regression and an array of research tools and models that can be utilized to advance this field. Additionally, we discuss the roles of lymphatic vessel regression and dysfunction in select pathologies, highlighting how an improved understanding of lymphatic vessel regression may yield therapeutic insights for these disease states.

Analyzing Vessel Regression and Endothelial Apoptosis as a Component of Angiogenic Vessel Remodeling.

Angiogenic vessel remodeling is a critical step in establishing a hierarchical vessel network. Vessel networks rapidly expand through angiogenesis in response to pro-angiogenic factors. This leads to an initially dense vessel network that requires selective regression of vessel branches to establish a hierarchical conduit for blood flow, a process known as pruning. This involves migration of endothelial cells from low-flow vessels to adjacent high-flow vessels and generally occurs independently of cell death. Vessels may also regress in response to other stimuli, including reduced metabolic demand, redundancy, and pathological stimuli. In these contexts, widespread vessel regression typically occurs and involves loss of endothelial cells by apoptotic cell death. Thus, vessel remodeling occurs via both apoptosis independent and dependent vessel regression. In this chapter, we outline a semi-automated method for quantifying vessel regression using the neonatal model of angiogenesis. We further provide instruction on analyzing endothelial apoptosis in this model.

Regression of Human Breast Carcinoma in Nude Mice after Adsflt Gene Therapy Is Mediated by Tumor Vascular Endothelial Cell Apoptosis.

Two vascular endothelial growth factor (VEGF) receptors, FLT-1 and KDR, are expressed preferentially in proliferating endothelium. There is increasing evidence that recombinant, soluble VEGF receptor domains interfering with VEGF signaling may inhibit in vivo neoangiogenesis, tumor growth and metastatic spread. We hypothesized that a soluble form of FLT-1 receptor (sFLT-1) could inhibit the growth of pre-established tumors via an anti-angiogenic mechanism. A replication-deficient adenovirus (Ad) vector carrying the sflt-1 cDNA (Adsflt) was used to overexpress the sFLT-1 receptor in a breast cancer animal model. MCF-7 cells, which produce VEGF, were used to establish solid tumors in the mammary fat pads of female nude mice. After six weeks, tumors were injected either with Adsflt or a negative control virus (AdCMV.ßgal). After six months, average tumor volume in the Adsflt-infected group (33 ± 22 mm3) decreased by 91% relative to that of the negative control group (388 ± 94 mm3; p < 0.05). Moreover, 10 of 15 Adsflt-infected tumors exhibited complete regression. The vascular density of Adsflt-infected tumors was reduced by 50% relative to that of negative controls (p < 0.05), which is consistent with sFLT-1-mediated tumor regression through an anti-angiogenic mechanism. Moreover, cell necrosis and fibrosis associated with long-term regression of Adsflt−infected tumors were preceded by apoptosis of tumor vascular endothelial cells. Mice treated with Adsflt intratumorally showed no delay in the healing of cutaneous wounds, providing preliminary evidence that Ad-mediated sFLT-1 overexpression may be an effective anti-angiogenic therapy for cancer without the risk of systemic anti-angiogenic effects.

BMP-SMAD1/5 Signaling Regulates Retinal Vascular Development.

Vascular development is an orchestrated process of vessel formation from pre-existing vessels via sprouting and intussusceptive angiogenesis as well as vascular remodeling to generate the mature vasculature. Bone morphogenetic protein (BMP) signaling via intracellular SMAD1 and SMAD5 effectors regulates sprouting angiogenesis in the early mouse embryo, but its role in other processes of vascular development and in other vascular beds remains incompletely understood. Here, we investigate the function of SMAD1/5 during early postnatal retinal vascular development using inducible, endothelium-specific deletion of Smad1 and Smad5. We observe the formation of arterial-venous malformations in areas with high blood flow, and fewer and less functional tip cells at the angiogenic front. The vascular plexus region is remarkably hyperdense and this is associated with reduced vessel regression and aberrant vascular loop formation. Taken together, our results highlight important functions of SMAD1/5 during vessel formation and remodeling in the early postnatal retina.

A Role for Low-Density Lipoprotein Receptor-Related Protein 6 in Blood Vessel Regression in Wound Healing.

Objective: The healing of skin wounds is typified by a pattern of robust angiogenesis followed by vascular regression. Pigment epithelium-derived factor (PEDF), a recognized endogenous antiangiogenic protein, regulates vascular regression in resolving wounds through an unknown receptor. Among the multiple receptors for PEDF that have been identified, low-density lipoprotein receptor-related protein 6 (Lrp6) has been described as a regulator of angiogenesis in multiple systems. The purpose of the current study was to determine if the Lrp6 receptor plays a role in vessel regression in wounds. Approach: Excisional skin wounds were prepared on C57BL/6 mice. RT-PCR and immunoblots were performed to measure Lrp6 expression over a time course of wound healing. Immunohistochemistry was performed to localize Lrp6 in both recombinant PEDF (rPEDF)-treated and control wounds. To examine whether Lrp6 is critical to the regulation of capillary regression in vivo, wounds were treated with Lrp6 siRNA to minimize its presence in wounds. Immunohistochemistry for CD31 was performed to quantify blood vessel density. Results: PCR and immunoblots revealed significant increases in Lrp6 expression during the vascular regression phase of wound healing. Lrp6 was found to colocalize with CD31+ endothelial cells in wounds. The addition of rPEDF to wounds caused an increase in Lrp6-CD31+ endothelial cell colocalization. Inhibition of Lrp6 by siRNA impeded the vascular regression phase of healing. Innovation: This study is the first to demonstrate an association between Lrp6 and vessel regression in wound healing. Conclusion: Lrp6 is expressed in wounds in a temporal and spatial manner that suggests it may be a receptor for PEDF during vascular regression. PEDF increases Lrp6 expression in the wound vasculature, and inhibition of Lrp6 blocked vascular regression in wounds. The results suggest that Lrp6 is important to vascular regression in wounds, possibly through direct interaction with PEDF.

Photoacoustic imaging for the evaluation of early tumor response to antivascular treatment.

BACKGROUND: Photoacoustic imaging (PAI) provides real-time noninvasive and contrast agent-free monitoring of the concentrations of some endogenous compounds related to tumor vascularization and oxygenation. In this study, we used PAI to noninvasively evaluate tumor responses to antiangiogenic therapy. METHODS: In vivo studies were performed with the approval of our institutional animal ethics committee. We used a xenograft mouse model of 4T1 breast cancer treated with different doses of bevacizumab or vehicle. Seven days after implantation, tumor-bearing mice (with tumors ~5-8 mm diameter) were randomly divided into low-dose (10 mg/kg), high-dose (20 mg/kg) and vehicle groups (same dose of saline). Each experimental group was administered bevacizumab intraperitoneally only once. Before and after treatment, acoustic resolution-photoacoustic microscopy (AR-PAM), a type of PAI, was conducted in vivo consecutively from day 1 to day 5. PAI-derived quantitative parameters were calculated at each time point. Additional cohorts of mice were used to quantify CD31 and hypoxia by immunohistochemical assays. RESULTS: The values of the PAI parameters were not significantly different among the experimental and control groups at the same time point before treatment (all P>0.05). The total hemoglobin (HbT) levels in the treatment group gradually decreased from day 1 to day 2 (relative to those in the control group, P>0.05) and decreased significantly relative to those in the control group from day 3 to day 5 (P<0.05). The deoxyhemoglobin (HbR) levels in the treatment group decreased from day 1 to 5 after treatment. The high-dose group had significantly decreased HbR levels relative to the control group from day 1 to 5 (P<0.05). The low-dose group also showed a gradual and significant decrease in HbR levels on day 3 (P<0.05). CD31 was decreased in the low-dose group relative to the control group on day 1 (decreased by 34.05%, P=0.067) and day 3 (decreased by 45.27%, P=0.180), and the decrease in CD31 persisted on day 5 (decreased by 71.41%, P=0.000). CD31 decreased to a greater extent in the high-dose group than in the low-dose group. Tumor hypoxia was significantly increased on day 1 from day 0 in the treatment groups (P<0.05), especially in the high-dose group. Hypoxia was decreased on days 3 and 5 in the low-dose group (10.92±0.92 and 8.17±1.9, P=0.317) but continuously increased over time in the high-dose group. Significantly greater hypoxia was observed in the high-dose group than in the low-dose group (17.60±1.20 and 20.33±0.47, P<0.05). CONCLUSIONS: PAI can be used to evaluate both vessel regression and hypoxia in response to anti-vascular treatment.

Stimulation and Inhibition of Lymphangiogenesis Via Adeno-Associated Viral Gene Delivery.

The lymphatic vessels can be selectively stimulated to grow in adult mice, rats and pigs by application of viral vectors expressing the lymphangiogenic factors VEGF-C or VEGF-D. Vice versa, lymphangiogenesis in various pathological settings can be inhibited by the blocking of the VEGF-C/VEGFR3 interaction using a ligand-binding soluble form of VEGFR3. Furthermore, the recently discovered plasticity of meningeal and lacteal lymphatic vessels provides novel opportunities for their manipulation in disease. Adenoviral and adeno-associated viral vectors (AAVs) provide suitable tools for establishing short- and long-term gene expression, respectively and adenoviral vectors have already been used in clinical trials. As an example, we describe here ways to manipulate the meningeal lymphatic vasculature in the adult mice via AAV-mediated gene delivery. The possibility of stimulation and inhibition of lymphangiogenesis in adult mice has enabled the analysis of the role and function of lymphatic vessels in mouse models of disease.

Non-canonical Wnt signalling modulates the endothelial shear stress flow sensor in vascular remodelling.

Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. Deregulation of endothelial responses to flow-induced shear is believed to contribute to many aspects of cardiovascular diseases including atherosclerosis. However, how molecular signals and shear-mediated physical forces integrate to regulate vascular patterning is poorly understood. Here we show that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/Wnt11 renders endothelial cells more sensitive to shear, resulting in axial polarization and migration against flow at lower shear levels. Integration of flow modelling and polarity analysis in entire vascular networks demonstrates that polarization against flow is achieved differentially in artery, vein, capillaries and the primitive sprouting front. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus.

Inflammation and glucose sensors: use of dexamethasone to extend glucose sensor function and life span in vivo.

BACKGROUND: It has been generally accepted that the acute loss of sensor function is the consequence of sensor biofouling as a result of inflammation induced at sites of sensor implantation, as well as tissue trauma induced by the sensor and its implantation. Because anti-inflammatory therapies are used routinely to control inflammation in a wide variety of diseases, we hypothesized that anti-inflammatory therapy would likely extend glucose sensor function in vivo. To test this hypothesis, we utilized our recently developed mouse model of implantable glucose sensors and the potent anti-inflammatory steroid dexamethasone (DEX). METHOD: For this study, glucose sensors were implanted subcutaneously into the head and neck area of mice and sensor function was determined up to 14 days postimplantation. These mice received a daily intraperitoneal injection of DEX at a dose of 1, 6, or 10 mg/kg body weight. RESULTS: Mice not treated with DEX lost sensor functionality very rapidly, usually within the first 24 hours postimplantation. Mice treated with DEX at the various doses had an increased sensor life span of up to 2 weeks postimplantation. Additionally, sensitivity was maintained in DEX-treated mice as compared to control mice (non-DEX treated). Histologic evaluation of tissue surrounding the site of sensor implantation had almost no inflammatory cells in DEX-treated mice, whereas control mice had an intense band of inflammation surrounding the site of sensor implantation. CONCLUSION: To our knowledge this is the first study directly demonstrating that anti-inflammatory therapy can extend glucose sensor function in vivo and supports the key role of inflammation in loss of sensor function in vivo, as well as the uses of anti-inflammatory therapy as a potential key adjuvant in enhancing glucose sensor function and life span in vivo.