Michael R. Mancuso

Inhibition of Vascular Endothelial Growth Factor (VEGF) Signaling in Cancer Causes Loss of Endothelial Fenestrations, Regression of Tumor Vessels, and Appearance of Basement Membrane Ghosts

Angiogenesis inhibitors are receiving increased attention as cancer therapeutics, but little is known of the cellular effects of these inhibitors on tumor vessels. We sought to determine whether two agents, AG013736 and VEGF-Trap, that inhibit vascular endothelial growth factor (VEGF) signaling, merely stop angiogenesis or cause regression of existing tumor vessels. Here, we report that treatment with these inhibitors caused robust and early changes in endothelial cells, pericytes, and basement membrane of vessels in spontaneous islet-cell tumors of RIP-Tag2 transgenic mice and in subcutaneously implanted Lewis lung carcinomas. Strikingly, within 24 hours, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and patency and blood flow ceased in some vessels. By 7 days, vascular density decreased more than 70%, and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells. Vessels in Lewis lung tumors, which lacked endothelial fenestrations, showed less regression. In both tumors, pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels acquired a more normal phenotype. Vascular basement membrane persisted after endothelial cells degenerated, providing a ghost-like record of pretreatment vessel number and location and a potential scaffold for vessel regrowth. The potent anti-vascular action observed is evidence that VEGF signaling inhibitors do more than stop angiogenesis. Early loss of endothelial fenestrations in RIP-Tag2 tumors is a clue that vessel phenotype may be predictive of exceptional sensitivity to these inhibitors.

Rapid vascular regrowth in tumors after reversal of VEGF inhibition

Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less alpha-SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.

Abnormalities of Basement Membrane on Blood Vessels and Endothelial Sprouts in Tumors

Often described as incomplete or absent, the basement membrane of blood vessels in tumors has attracted renewed attention as a source of angiogenic and anti-angiogenic molecules, site of growth factor binding, participant in angiogenesis, and potential target in cancer therapy. This study evaluated the composition, extent, and structural integrity of the basement membrane on blood vessels in three mouse tumor models: spontaneous RIP-Tag2 pancreatic islet tumors, MCa-IV mammary carcinomas, and Lewis lung carcinomas. Tumor vessels were identified by immunohistochemical staining for the endothelial cell markers CD31, endoglin (CD105), vascular endothelial growth factor receptor-2, and integrin alpha5 (CD49e). Confocal microscopic studies revealed that basement membrane identified by type IV collagen immunoreactivity covered >99.9% of the surface of blood vessels in the three tumors, just as in normal pancreatic islets. Laminin, entactin/nidogen, and fibronectin immunoreactivities were similarly ubiquitous on tumor vessels. Holes in the basement membrane, found by analyzing 1- micro m confocal optical sections, were <2.5 micro m in diameter and involved only 0.03% of the vessel surface. Despite the extensive vessel coverage, the basement membrane had conspicuous structural abnormalities, including a loose association with endothelial cells and pericytes, broad extensions away from the vessel wall, and multiple layers visible by electron microscopy. Type IV collagen-immunoreactive sleeves were also present on endothelial sprouts, supporting the idea that basement membrane is present where sprouts grow and regress. These findings indicate that basement membrane covers most tumor vessels but has profound structural abnormalities, consistent with the dynamic nature of endothelial cells and pericytes in tumors.

Attribution of vascular phenotypes of the murine Egfl7 locus to the microRNA miR-126

Intronic microRNAs have been proposed to complicate the design and interpretation of mouse knockout studies. The endothelial-expressed Egfl7/miR-126 locus contains miR-126 within Egfl7 intron 7, and angiogenesis deficits have been previously ascribed to Egfl7 gene-trap and lacZ knock-in mice. Surprisingly, selectively floxed Egfl7Δ and miR-126Δ alleles revealed that Egfl7Δ/Δ mice were phenotypically normal, whereas miR-126Δ/Δ mice bearing a 289-nt microdeletion recapitulated previously described Egfl7 embryonic and postnatal retinal vascular phenotypes. Regulation of angiogenesis by miR-126 was confirmed by endothelial-specific deletion and in the adult cornea micropocket assay. Furthermore, miR-126 deletion inhibited VEGF-dependent Akt and Erk signaling by derepression of the p85β subunit of PI3 kinase and of Spred1, respectively. These studies demonstrate the regulation of angiogenesis by an endothelial miRNA, attribute previously described Egfl7 vascular phenotypes to miR-126, and document inadvertent miRNA dysregulation as a complication of mouse knockout strategies.

Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124

Plumbing in the Brain Superficial similarities of vasculature in different parts of the body may mask organ-specific developmental nuances. The vasculature of the brain uniquely has to insulate the organ from insults that the rest of the body must tolerate. Kuhnert et al. (p. 985) analyzed the developmental uniqueness of the brains vasculature through study of a G protein–coupled receptor, GPR124, initially identified by its actions in the vasculature of colon cancer. GPR124 is also involved in normal development of the brains vasculature. Mice expressing low levels of GPR124 did not develop adequate vasculature in the brain and died from hemorrhages. Mice with too much GPR124 developed a tangled, thin-walled, excessive vasculature in the brain. Although the overexpressing mice survived, they were prone to neurological symptoms such as ataxia. GPR124 seems to control the normal development of the endothelial cells, particularly in the forebrain and ventral neural tube. A factor is identified that determines the amount of vasculature in the brain, and, in doing so, affects brain function. The orphan G protein–coupled receptor (GPCR) GPR124/tumor endothelial marker 5 is highly expressed in central nervous system (CNS) endothelium. Here, we show that complete null or endothelial-specific GPR124 deletion resulted in embryonic lethality from CNS-specific angiogenesis arrest in forebrain and neural tube. Conversely, GPR124 overexpression throughout all adult vascular beds produced CNS-specific hyperproliferative vascular malformations. In vivo, GPR124 functioned cell-autonomously in endothelium to regulate sprouting, migration, and developmental expression of the blood-brain barrier marker Glut1, whereas in vitro, GPR124 mediated Cdc42-dependent directional migration to forebrain-derived, vascular endothelial growth factor–independent cues. Our results demonstrate CNS-specific angiogenesis regulation by an endothelial receptor and illuminate functions of the poorly understood adhesion GPCR subfamily. Further, the functional tropism of GPR124 marks this receptor as a therapeutic target for CNS-related vascular pathologies.

Cellular Source and Amount of Vascular Endothelial Growth Factor and Platelet-Derived Growth Factor in Tumors Determine Response to Angiogenesis Inhibitors

Vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and their receptors are important targets in cancer therapy based on angiogenesis inhibition. However, it is unclear whether inhibition of VEGF and PDGF together is more effective than inhibition of either one alone. Here, we used two contrasting tumor models to compare the effects of inhibiting VEGF or PDGF alone, by adenovirally generated soluble receptors, to the effects of inhibiting both together. In RIP-Tag2 tumors, VEGF and PDGF inhibition together reduced tumor vascularity and abundance of pericytes. However, VEGF inhibition reduced tumor vascularity without decreasing pericyte density, and PDGF inhibition reduced pericytes without reducing tumor vascularity. By contrast, in Lewis lung carcinomas (LLC), inhibition of VEGF or PDGF reduced blood vessels and pericytes to the same extent as did inhibition of both together. Similar results were obtained using tyrosine kinase inhibitors AG-013736 and imatinib. In LLC, VEGF expression was largely restricted to pericytes and PDGF was largely restricted to endothelial cells, but, in RIP-Tag2 tumors, expression of both growth factors was more widespread and significantly greater than in LLC. These findings suggest that inhibition of PDGF in LLC reduced pericytes, and then tumor vessels regressed because pericytes were the main source of VEGF. The vasculature of RIP-Tag2 tumors, in which most VEGF is from tumor cells, was more resistant to PDGF inhibition. The findings emphasize the interdependence of pericytes and endothelial cells in tumors and the importance of tumor phenotype in determining the cellular effects of VEGF and PDGF inhibitors on tumor vessels.

Developmental Angiogenesis of the Central Nervous System

The vasculature of the central nervous system (CNS) is highly specialized with a blood-brain-barrier, reciprocal neuroepithelial-endothelial cell interactions and extensive pericyte coverage. Developmentally, numerous important signaling pathways participate in CNS angiogenesis to orchestrate the precise timing and spatial arrangement of the complex CNS vascular network. From a therapeutic standpoint, the CNS vasculature has attracted increased attention since many human ailments, such as stroke, retinopathy, cancer and autoimmune disease are intimately associated with the biology of CNS blood vessels. This review focuses on growth factor pathways that have been shown to be important in developmental CNS vascularization through studies of mouse genetic models and human diseases.

Gpr124 is essential for blood–brain barrier integrity in central nervous system disease

Although blood–brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt–β-catenin signaling. Constitutive activation of Wnt–β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

Endocrine therapy and strategies to overcome therapeutic resistance in breast cancer

Despite the remarkable success of endocrine therapy in the treatment of patients with estrogen receptor (ER)- positive breast cancer, not all patients derive benefit from such therapy, or may benefit only temporarily before disease progression or relapse occurs. The value of endocrine therapy, which blocks ER signaling by a variety of strategies, lies in its simplicity, lower toxicity, and better alignment with preserved quality of life, particularly when compared to chemotherapy, which is more toxic and has only modest benefits for many patients with ER-positive breast cancer. It is therefore critical that we discover ways to extend endocrine therapy benefit in patients and prevent therapeutic resistance whenever possible. The tremendous evolution in our understanding of endocrine resistance mechanisms, coupled with the increasing availability of novel agents that target resistance pathways, has led to enhanced treatment approaches for patients with ER-positive breast cancer, primarily through combinations of endocrine agents with a variety of pathway inhibitors. Despite these treatment advances and our changing view of ER-positive breast cancer, there is much work that needs to be done. It remains a problem that we cannot reliably predict which subsets of patients will experience disease relapse or progression on endocrine therapy, and as such, combination strategies with targeted agents have largely been used in unselected patients with ER-positive breast cancer, including those who continue to have endocrine-sensitive disease. Patient selection is a significant issue since most of the targeted therapeutics that we use with endocrine therapy are expensive and can be toxic, and we may be inadvertently overtreating patients whose disease can still be controlled with endocrine therapy alone. In this article, we will review current and future strategies in the treatment of ER-positive breast cancer, as well as the evolving role of targeted therapy in the management of endocrine-resistance.

Signaling in Normal and Pathological Angiogenesis

Angiogenesis, the growth of new blood vessels from pre-existing ones, is crucial for the growth and development of many multicellular organisms and for the progression of many diseases such as cancer, macular degeneration, and diabetic retinopathy. The process of angiogenesis begins with a combination of highly controlled endothelial cell migration (tip cell phenotype) and proliferation (stalk cell phenotype) to form new immature blood vessels. These newly formed blood vessels, also termed “neovessels,” are then stabilized through recruitment of mural cells to generate a mature vascular network that is required for the metabolic demands of the underlying tissue, and provides a conduit through which the immune system functions. A delicate balance between positive and negative angiogenic regulators governs this complex process. In this chapter we will focus on the key signaling events that regulate angiogenesis both during development and in disease.