Frank Kuhnert

Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1

Whereas the adult gastrointestinal epithelium undergoes tremendous self-renewal through active proliferation in crypt stem cell compartments, the responsible growth factors regulating this continuous proliferation have not been defined. The exploration of physiologic functions of Wnt proteins in adult organisms has been hampered by functional redundancy and the necessity for conditional inactivation strategies. Dickkopf-1 (Dkk1) is a potent secreted Wnt antagonist that interacts with Wnt coreceptors of the LRP family. To address the contribution of Wnt signaling to gastrointestinal epithelial proliferation, adenoviral expression of Dkk1 was used to achieve stringent, conditional, and reversible Wnt inhibition in adult animals. Adenovirus Dkk1 (Ad Dkk1) treatment of adult mice repressed expression of the Wnt target genes CD44 and EphB2 within 2 days in both small intestine and colon, indicating an extremely broad role for Wnt signaling in the maintenance of adult gastrointestinal gene expression. In parallel, Ad Dkk1 markedly inhibited proliferation in small intestine and colon, accompanied by progressive architectural degeneration with the loss of crypts, villi, and glandular structure by 7 days. Whereas decreased Dkk1 expression at later time points (>10 days) was followed by crypt and villus regeneration, which was consistent with a reversible process, substantial mortality ensued from colitis and systemic infection. These results indicate the efficacy of systemic expression of secreted Wnt antagonists as a general strategy for conditional inactivation of Wnt signaling in adult organisms and illustrate a striking reliance on a single growth factor pathway for the maintenance of the architecture of the adult small intestine and colon.

Wnt/β-catenin signaling is required for CNS, but not non-CNS, angiogenesis

Despite the importance of CNS blood vessels, the molecular mechanisms that regulate CNS angiogenesis and blood−brain barrier (BBB) formation are largely unknown. Here we analyze the role of Wnt/β-catenin signaling in regulating the formation of CNS blood vessels. First, through the analysis of TOP-Gal Wnt reporter mice, we identify that canonical Wnt/β-catenin signaling is specifically activated in CNS, but not non-CNS, blood vessels during development. This activation correlates with the expression of different Wnt ligands by neural progenitor cells in distinct locations throughout the CNS, including Wnt7a and Wnt7b in ventral regions and Wnt1, Wnt3, Wnt3a, and Wnt4 in dorsal regions. Blockade of Wnt/β-catenin signaling in vivo specifically disrupts CNS, but not non-CNS, angiogenesis. These defects include reduction in vessel number, loss of capillary beds, and the formation of hemorrhagic vascular malformations that remain adherent to the meninges. Furthermore, we demonstrate that Wnt/β-catenin signaling regulates the expression of the BBB-specific glucose transporter glut-1. Taken together these experiments reveal an essential role for Wnt/β-catenin signaling in driving CNS-specific angiogenesis and provide molecular evidence that angiogenesis and BBB formation are in part linked.

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.

Targeting Endothelium-Pericyte Cross Talk by Inhibiting VEGF Receptor Signaling Attenuates Kidney Microvascular Rarefaction and Fibrosis

Microvascular pericytes and perivascular fibroblasts have recently been identified as the source of scar-producing myofibroblasts that appear after injury of the kidney. We show that cross talk between pericytes and endothelial cells concomitantly dictates development of fibrosis and loss of microvasculature after injury. When either platelet-derived growth factor receptor (R)-β signaling in pericytes or vascular endothelial growth factor (VEGF)R2 signaling in endothelial cells was blocked by circulating soluble receptor ectodomains, both fibrosis and capillary rarefaction were markedly attenuated during progressive kidney injury. Blockade of either receptor-mediated signaling pathway prevented pericyte differentiation and proliferation, but VEGFR2 blockade also attenuated recruitment of inflammatory macrophages throughout disease progression. Whereas injury down-regulated angiogenic VEGF164, the dys-angiogenic isomers VEGF120 and VEGF188 were up-regulated, suggesting that pericyte-myofibroblast differentiation triggers endothelial loss by a switch in secretion of VEGF isomers. These findings link fibrogenesis inextricably with microvascular rarefaction for the first time, add new significance to fibrogenesis, and identify novel therapeutic targets.

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.

Soluble receptor-mediated selective inhibition of VEGFR and PDGFRβ signaling during physiologic and tumor angiogenesis

The simultaneous targeting of both endothelial cells and pericytes via inhibition of VEGF receptor (VEGFR) and PDGFβ receptor (PDGFRβ) signaling, respectively, has been proposed to enhance the efficacy of antiangiogenic tumor therapy. Clinical and preclinical modeling of combined VEGFR and PDGFRβ signaling inhibition, however, has used small molecule kinase inhibitors with inherently broad substrate specificities, precluding detailed examination of this hypothesis. Here, adenoviral expression of a soluble VEGFR2/Flk1 ectodomain (Ad Flk1-Fc) in combination with a soluble ectodomain of PDGFRβ (Ad sPDGFRβ) allowed highly selective inhibition of these pathways. The activity of Ad sPDGFRβ was validated in vitro against PDGF-BB and in vivo with near-complete blockade of pericyte recruitment in the angiogenic corpus luteum, resulting in prominent hemorrhage, thus demonstrating an essential function for PDGF signaling during ovarian angiogenesis. Combination therapy with Ad PDGFRβ and submaximal doses of Ad Flk1-Fc produced modest additive antitumor effects; however, no additivity was observed with maximal VEGF inhibition in numerous s.c. models. Notably, VEGF inhibition via Ad Flk1-Fc was sufficient to strongly suppress tumor endothelial and pericyte content as well as intratumoral PDGF-B mRNA, obscuring additive Ad sPDGFRβ effects on pericytes or tumor volume. These studies using highly specific soluble receptors suggest that additivity between VEGFR and PDGFRβ inhibition depends on the strength of VEGF blockade and appears minimal under conditions of maximal VEGF antagonism.

Reversible cell-cycle entry in adult kidney podocytes through regulated control of telomerase and Wnt signaling

Mechanisms of epithelial cell renewal remain poorly understood in the mammalian kidney, particularly in the glomerulus, a site of cellular damage in chronic kidney disease. Within the glomerulus, podocytes—differentiated epithelial cells crucial for filtration—are thought to lack substantial capacity for regeneration. Here we show that podocytes rapidly lose differentiation markers and enter the cell cycle in adult mice in which the telomerase protein component TERT is conditionally expressed. Transgenic TERT expression in mice induces marked upregulation of Wnt signaling and disrupts glomerular structure, resulting in a collapsing glomerulopathy resembling those in human disease, including HIV-associated nephropathy (HIVAN). Human and mouse HIVAN kidneys show increased expression of TERT and activation of Wnt signaling, indicating that these are general features of collapsing glomerulopathies. Silencing transgenic TERT expression or inhibiting Wnt signaling through systemic expression of the Wnt inhibitor Dkk1 in either TERT transgenic mice or in a mouse model of HIVAN results in marked normalization of podocytes, including rapid cell-cycle exit, re-expression of differentiation markers and improved filtration barrier function. These data reveal an unexpected capacity of podocytes to reversibly enter the cell cycle, suggest that podocyte renewal may contribute to glomerular homeostasis and implicate the telomerase and Wnt–β-catenin pathways in podocyte proliferation and disease.

VEGF signaling has distinct spatiotemporal roles during heart valve development.

Heart valve malformations are one of the most common types of birth defects, illustrating the complex nature of valve development. Vascular endothelial growth factor (VEGF) signaling is one pathway implicated in valve formation, however its specific spatial and temporal roles remain poorly defined. To decipher these contributions, we use two inducible dominant negative approaches in mice to disrupt VEGF signaling at different stages of embryogenesis. At an early step in valve development, VEGF signals are required for the full transformation of endocardial cells to mesenchymal cells (EMT) at the outflow tract (OFT) but not atrioventricular canal (AVC) endocardial cushions. This role likely involves signaling mediated by VEGF receptor 1 (VEGFR1), which is highly expressed in early cushion endocardium before becoming downregulated after EMT. In contrast, VEGFR2 does not exhibit robust cushion endocardium expression until after EMT is complete. At this point, VEGF signaling acts through VEGFR2 to direct the morphogenesis of the AVC cushions into mature, elongated valve leaflets. This latter role of VEGF requires the VEGF-modulating microRNA, miR-126. Thus, VEGF roles in the developing valves are dynamic, transitioning from a differentiation role directed by VEGFR1 in the OFT to a morphogenetic role through VEGFR2 primarily in the AVC-derived valves.

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.