Guo-Hua Fong

Placental but Not Heart Defects Are Associated with Elevated Hypoxia-Inducible Factor α Levels in Mice Lacking Prolyl Hydroxylase Domain Protein 2

ABSTRACT PHD1, PHD2, and PHD3 are prolyl hydroxylase domain proteins that regulate the stability of hypoxia-inducible factor α subunits (HIF-α). To determine the roles of individual PHDs during mouse development, we disrupted all three Phd genes and found that Phd2−/− embryos died between embryonic days 12.5 and 14.5 whereas Phd1−/− or Phd3−/− mice were apparently normal. In Phd2−/− mice, severe placental and heart defects preceded embryonic death. Placental defects included significantly reduced labyrinthine branching morphogenesis, widespread penetration of the labyrinth by spongiotrophoblasts, and abnormal distribution of trophoblast giant cells. The expression of several trophoblast markers was also altered, including an increase in the spongiotrophoblast marker Mash2 and decreases in the labyrinthine markers Tfeb and Gcm1. In the heart, trabeculae were poorly developed, the myocardium was remarkably thinner, and interventricular septum was incompletely formed. Surprisingly, while there were significant global increases in HIF-α protein levels in the placenta and the embryo proper, there was no specific HIF-α increase in the heart. Taken together, these data indicate that among all three PHD proteins, PHD2 is uniquely essential during mouse embryogenesis.

A hierarchical order of factors in the generation of FLK1- and SCL-expressing hematopoietic and endothelial progenitors from embryonic stem cells

The receptor tyrosine kinase FLK1 and the transcription factor SCL play crucial roles in the establishment of hematopoietic and endothelial cell lineages in mice. We have previously used an in vitro differentiation model of embryonic stem (ES) cells and demonstrated that hematopoietic and endothelial cells develop via sequentially generated FLK1+ and SCL+ cells. To gain a better understanding of cellular and molecular events leading to hematopoietic specification, we examined factors necessary for FLK1+ and SCL+ cell induction in serum-free conditions. We demonstrate that bone morphogenetic protein (BMP) 4 was required for the generation of FLK1+ and SCL+ cells, and that vascular endothelial growth factor (VEGF) was necessary for the expansion and differentiation of SCL-expressing hematopoietic progenitors. Consistently, Flk1-deficient ES cells responded to BMP4 and generated TER119+ and CD31+ cells, but they failed to expand in response to VEGF. The Smad1/5 and map kinase pathways were activated by BMP4 and VEGF, respectively. The overexpression of SMAD6 in ES cells resulted in a reduction of FLK1+ cells. In addition, a MAP kinase kinase 1 specific inhibitor blocked the expansion of SCL+ cells in response to VEGF. Finally, VEGF mediated expansion of hematopoietic and endothelial cell progenitors was inhibited by TGFβ1, but was augmented by activin A. Our studies suggest that hematopoietic and endothelial commitment from the mesoderm occurs via BMP4-mediated signals and that expansion and/or differentiation of such progenitors is achieved by an interplay of VEGF, TGFβ1 and activin A signaling.

Role and regulation of prolyl hydroxylase domain proteins

Oxygen-dependent hydroxylation of hypoxia-inducible factor (HIF)-α subunits by prolyl hydroxylase domain (PHD) proteins signals their polyubiquitination and proteasomal degradation, and plays a critical role in regulating HIF abundance and oxygen homeostasis. While oxygen concentration plays a major role in determining the efficiency of PHD-catalyzed hydroxylation reactions, many other environmental and intracellular factors also significantly modulate PHD activities. In addition, PHDs may also employ hydroxylase-independent mechanisms to modify HIF activity. Interestingly, while PHDs regulate HIF-α protein stability, PHD2 and PHD3 themselves are subject to feedback upregulation by HIFs. Functionally, different PHD isoforms may differentially contribute to specific pathophysiological processes, including angiogenesis, erythropoiesis, tumorigenesis, and cell growth, differentiation and survival. Because of diverse roles of PHDs in many different processes, loss of PHD expression or function triggers multi-faceted pathophysiological changes as has been shown in mice lacking different PHD isoforms. Future investigations are needed to explore in vivo specificity of PHDs over different HIF-α subunits and differential roles of PHD isoforms in different biological processes.

Essential Role for Prolyl Hydroxylase Domain Protein 2 in Oxygen Homeostasis of the Adult Vascular System

Background— Prolyl hydroxylase domain (PHD) proteins, including PHD1, PHD2, and PHD3, mediate oxygen-dependent degradation of hypoxia-inducible factor (HIF)-α subunits. Although angiogenic roles of hypoxia-inducible factors are well known, the roles of PHDs in the vascular system remain to be established. Methods and Results— We evaluated angiogenic phenotypes in mice carrying targeted disruptions in genes encoding different PHD isoforms. Although Phd1−/− and Phd3−/− mice did not display apparent angiogenic defects, broad-spectrum conditional knockout of Phd2 led to hyperactive angiogenesis and angiectasia. Blood vessels in PHD2-deficient mice were highly perfusable. Furthermore, examination of medium-sized vessels in subendocardial layer in the heart demonstrated successful recruitment of vascular smooth muscle cells. Surprisingly, increased vascular growth was independent of local efficiency of Phd2 disruption. Mice carrying significant Phd2 disruption in multiple organs, including the liver, heart, kidney, and lung, displayed excessive vascular growth not only in these organs but also in the brain, where Phd2 disruption was very inefficient. More surprisingly, increased accumulation of hypoxia-inducible factor-1α and angiectasia in the liver were not accompanied by corresponding increases in hepatic expression of Vegfa or angiopoietin-1. However, the serum vascular endothelial growth factor-A level was significantly increased in PHD2-deficient mice. Conclusions— PHD2, but not PHD1 and PHD3, is a major negative regulator for vascular growth in adult mice. Increased angiogenesis in PHD2-deficient mice may be mediated by a novel systemic mechanism.

VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis

VEGF-B, a homolog of VEGF discovered a long time ago, has not been considered an important target in antiangiogenic therapy. Instead, it has received little attention from the field. In this study, using different animal models and multiple types of vascular cells, we revealed that although VEGF-B is dispensable for blood vessel growth, it is critical for their survival. Importantly, the survival effect of VEGF-B is not only on vascular endothelial cells, but also on pericytes, smooth muscle cells, and vascular stem/progenitor cells. In vivo, VEGF-B targeting inhibited both choroidal and retinal neovascularization. Mechanistically, we found that the vascular survival effect of VEGF-B is achieved by regulating the expression of many vascular prosurvival genes via both NP-1 and VEGFR-1. Our work thus indicates that the function of VEGF-B in the vascular system is to act as a “survival,” rather than an “angiogenic” factor and that VEGF-B inhibition may offer new therapeutic opportunities to treat neovascular diseases.

VEGF-B inhibits apoptosis via VEGFR-1–mediated suppression of the expression of BH3-only protein genes in mice and rats

Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. Using gene expression profiling of mouse primary aortic smooth muscle cells, and confirming the results by real-time PCR using mouse and rat cell lines, we showed that VEGF-B inhibited the expression of genes encoding the proapoptotic BH3-only proteins and other apoptosis- and cell death-related proteins, including p53 and members of the caspase family, via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Interestingly, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization, suggesting that VEGF-B is the first member of the VEGF family that has a potent antiapoptotic effect while lacking a general angiogenic activity. These findings indicate that VEGF-B may potentially offer a new therapeutic option for the treatment of neurodegenerative diseases.

Mechanisms of adaptive angiogenesis to tissue hypoxia.

Angiogenesis is mostly an adaptive response to tissue hypoxia, which occurs under a wide variety of situations ranging from embryonic development to tumor growth. In general, angiogenesis is dependent on the accumulation of hypoxia inducible factors (HIFs), which are heterodimeric transcription factors of α and β subunits. Under normoxia, HIF heterodimers are not abundantly present due to oxygen dependent hydroxylation, polyubiquitination, and proteasomal degradation of α subunits. Under hypoxia, however, α subunits are stabilized and form heterodimers with HIF-1β which is not subject to oxygen dependent regulation. The accumulation of HIFs under hypoxia allows them to activate the expression of many angiogenic genes and therefore initiates the angiogenic process. In recent years, however, it has become clear that various other mechanisms also participate in fine tuning angiogenesis. In this review, I discuss the relationship between hypoxia and angiogenesis under five topics: (1) regulation of HIF-α abundance and activity by oxygen tension and other conditions including oxygen independent mechanisms; (2) hypoxia-regulated expression of angiogenic molecules by HIFs and other transcription factors; (3) responses of vascular cells to hypoxia; (4) angiogenic phenotypes due to altered HIF signaling in mice; and (5) role of the HIF pathway in pathological angiogenesis. Studies discussed under these topics clearly indicate that while mechanisms of oxygen-regulated HIF-α stability provide exciting opportunities for the development of angiogenesis or anti-angiogenesis therapies, it is also highly important to consider various other mechanisms for the optimization of these procedures.

Regulation of flt‐1 expression during mouse embryogenesis suggests a role in the establishment of vascular endothelium

Flt‐1 is a high affinity binding receptor for the vascular endothelial cell growth factor (VEGF) and is primarily expressed in endothelial cells. In this study we have investigated the temporal and spatial regulation of its expression by establishing mouse lines containing the lacZ gene targeted into the flt‐1 locus through homologous recombination in embryonic stem (ES) cells. In the yolk sac as well as in the embryo proper, lacZ expression faithfully reflected the endogenous expression pattern of the flt‐1 gene. LacZ staining of heterozygous embryos led to the following observations: (1) the onset of flt‐1 expression is detected at the early primitive streak stage in the extraembryonic mesoderm, and is strongly up‐regulated thereafter, reaching a maximum by early to midsomite stages and declining subsequently; (2) while flt‐1 is widely expressed within the developing vascular endothelium, its expression level is differentially regulated both spatially and temporally. The pattern of flt‐1 expression suggests that it may play an important role in the initiation of endothelium development; and (3) flt‐1 is expressed in essentially all the cells in early blood islands, but later its expression is gradually restricted to the endothelial lineage. Our results indicate that flt‐1 is a marker for hemangioblasts, the presumed progenitor for both hematopoietic and angioblastic lineage. The flt‐1 expression pattern also suggests that it may play important roles in both vasculogenesis and angiogenesis.

Regulation of angiogenesis by oxygen sensing mechanisms

The choices for blood vessels to undergo angiogenesis or stay quiescent are mostly determined by the status of tissue oxygenation. A major link between tissue hypoxia and active angiogenesis is the accumulation of hypoxia-inducible factor (HIF)-α subunits which play a major role in the transcriptional activation of genes encoding angiogenic factors. HIF-α abundance is negatively regulated by a subfamily of dioxygenases referred to as prolyl hydroxylase domain-containing proteins (PHDs) which use O2 as a substrate to hydroxylate HIF-α subunits and hence tag them for rapid degradation. Under hypoxic conditions, HIF-α subunits accumulate due to reduced hydroxylation efficiency and form transcriptionally active heterodimers with HIF-1ß to activate the expression of angiogenic factors and other proteins important for cellular adaptation to hypoxia. Angiogenesis is regulated by a combination of at least two different mechanisms. The paracrine mechanism is mediated by non-endothelial expression of angiogenic factors such as vascular endothelial growth factor (VEGF)-A, which in turn interact with endothelial cell surface receptors to initiate angiogenic activities. In the autocrine mechanism, endothelial cell themselves are induced to express VEGF-A, which collaborate with the paracrine mechanism to support angiogenesis and protect vascular integrity. Because of critical roles of PHDs and HIFs in regulating angiogenic activities, studies are underway to assess their candidacy as targets for angiogenesis therapies.

VEGFR1 Tyrosine Kinase Signaling Promotes Lymphangiogenesis as Well as Angiogenesis Indirectly via Macrophage Recruitment

Objective—Angiogenesis and lymphangiogenesis are complex phenomena that involve the interplay of several growth factors and receptors. Recently, we have demonstrated that in Keratin-14 (K14) promoter-driven Vegf-A transgenic (Tg) mice, not only angiogenesis but also lymphangiogenesis is stimulated. However, the mechanism by which VEGFR1 is involved in lymphangiogenesis remains unclear. Methods and Results—To examine how important the tyrosine kinase (TK) of VEGFR1 is in lymphangiogenesis in K14 Vegf-A Tg mice, we crossed the K14 Vegf-A Tg mice with VEGFR1-TK–deficient mice to generate double mutant K14 Vegf-A Tg Vegfr1 tk−/− mice. K14 Vegf-A Tg Vegfr1 tk−/− mice exhibit a remarkable decrease in lymphangiogensis as well as angiogenesis in subcutaneous tissues. To address the mechanism underlying the decrease in lymphangiogensis, we investigated the recruitment of monocyte-macrophage-lineage cells into the skin. The recruitment of VEGFR1-expressing macrophages driven by VEGF-A was reduced in K14 Vegf-A Tg Vegfr1 tk−/− mice. Vegf-A Tg mice that received VEGFR1-TK–deficient bone marrow showed a reduction of macrophage recruitment, lymphangiogenesis and angiogenesis compared with those in K14 Vegf-A Tg mice. Conclusions—VEGFR1 signaling promotes lymphangiogenesis as well as angiogenesis mainly by increasing bone marrow–derived macrophage recruitment.