Masatsugu Ema

Neurons Limit Angiogenesis by Titrating VEGF in Retina

Vascular and nervous systems, two major networks in mammalian bodies, show a high degree of anatomical parallelism and functional crosstalk. During development, neurons guide and attract blood vessels, and consequently this parallelism is established. Here, we identified a noncanonical neurovascular interaction in eye development and disease. VEGFR2, a critical endothelial receptor for VEGF, was more abundantly expressed in retinal neurons than in endothelial cells, including endothelial tip cells. Genetic deletion of VEGFR2 in neurons caused misdirected angiogenesis toward neurons, resulting in abnormally increased vascular density around neurons. Further genetic experiments revealed that this misdirected angiogenesis was attributable to an excessive amount of VEGF protein around neurons caused by insufficient engulfment of VEGF by VEGFR2-deficient neurons. Moreover, absence of neuronal VEGFR2 caused misdirected regenerative angiogenesis in ischemic retinopathy. Thus, this study revealed neurovascular crosstalk and unprecedented cellular regulation of VEGF: retinal neurons titrate VEGF to limit neuronal vascularization. PAPERFLICK:

Platelet demand modulates the type of intravascular protrusion of megakaryocytes in bone marrow

Megakaryocytes (MKs) generate platelets via intravascular protrusions termed proplatelets, which are tandem arrays of platelet-sized swellings with a beaded appearance. However, it remains unclear whether all intravascular protrusions in fact become proplatelets, and whether MKs generate platelets without forming proplatelets. Here, we visualised the sequential phases of intravascular MK protrusions and fragments in living mouse bone marrow (BM), using intravital microscopy, and examined their ultrastructure. The formation of intravascular protrusions was observed to be a highly dynamic process, in which the size and shape of the protrusions changed sequentially prior to the release of platelet progenitors. Among these intravascular protrusions, immature thick protrusions were distinguished from proplatelets by their size and the dynamic morphogenesis seen by time-lapse observation. In ultrastructural analyses, the thick protrusions and their fragments were characterised by a peripheral zone, abundant endoplasmic reticulum and demarcation membrane system, and random microtubule arrays. Proplatelets were predominant among BM sinusoids in the physiological state; however, during an acute thrombocytopenic period, thick protrusions increased markedly in the sinusoids. These results strongly suggested that BM MKs form and release two types of platelet progenitors via distinct intravascular protrusions, and that platelet demand modulates the type of intravascular protrusion that is formed in vivo.

Developmental regression of hyaloid vasculature is triggered by neurons

Kubota and colleagues show that neurons sequester VEGF to prune blood vessels during eye development.

Osteogenic capillaries orchestrate growth plate-independent ossification of the malleus

Endochondral ossification is a developmental process by which cartilage is replaced by bone. Terminally differentiated hypertrophic chondrocytes are calcified, vascularized, and removed by chondroclasts before bone matrix is laid down by osteoblasts. In mammals, the malleus is one of three auditory ossicles that transmit vibrations of the tympanic membrane to the inner ear. The malleus is formed from a cartilaginous precursor without growth plate involvement, but little is known about how bones of this type undergo endochondral ossification. Here, we demonstrate that in the processus brevis of the malleus, clusters of osteoblasts surrounding the capillary loop produce bone matrix, causing the volume of the capillary lumen to decrease rapidly in post-weaning mice. Synchrotron X-ray tomographic microscopy revealed a concentric, cylindrical arrangement of osteocyte lacunae along capillaries, indicative of pericapillary bone formation. Moreover, we report that overexpression of Fosl1, which encodes a component of the AP-1 transcription factor complex, in osteoblasts significantly blocked malleal capillary narrowing. These data suggest that osteoblast/endothelial cell interactions control growth plate-free endochondral ossification through ‘osteogenic capillaries’ in a Fosl1-regulated manner. Summary: The endochondral ossification of the malleus, an ossicle of the mouse inner ear, occurs around capillaries and is mediated by the AP-1 transcription factor Fosl1.

Prox1-GFP/Flt1-DsRed transgenic mice: an animal model for simultaneous live imaging of angiogenesis and lymphangiogenesis

The roles of angiogenesis in development, health, and disease have been studied extensively; however, the studies related to lymphatic system are limited due to the difficulty in observing colorless lymphatic vessels. But recently, with the improved technique, the relative importance of the lymphatic system is just being revealed. We bred transgenic mice in which lymphatic endothelial cells express GFP (Prox1-GFP) with mice in which vascular endothelial cells express DsRed (Flt1-DsRed) to generate Prox1-GFP/Flt1-DsRed (PGFD) mice. The inherent fluorescence of blood and lymphatic vessels allows for direct visualization of blood and lymphatic vessels in various organs via confocal and two-photon microscopy and the formation, branching, and regression of both vessel types in the same live mouse cornea throughout an experimental time course. PGFD mice were bred with CDh5CreERT2 and VEGFR2lox knockout mice to examine specific knockouts. These studies showed a novel role for vascular endothelial cell VEGFR2 in regulating VEGFC-induced corneal lymphangiogenesis. Conditional deletion of vascular endothelial VEGFR2 abolished VEGFA- and VEGFC-induced corneal lymphangiogenesis. These results demonstrate the potential use of the PGFD mouse as a powerful animal model for studying angiogenesis and lymphangiogenesis.

Neural retina-specific Aldh1a1 controls dorsal choroidal vascular development via Sox9 expression in retinal pigment epithelial cells

VEGF secreted from retinal pigment epithelial (RPE) cells is responsible for the choroidal vascular development; however, the molecular regulatory mechanism is unclear. We found that Aldh1a1–/– mice showed choroidal hypoplasia with insufficient vascularization in the dorsal region, although Aldh1a1, an enzyme that synthesizes retinoic acids (RAs), is expressed in the dorsal neural retina, not in the RPE/choroid complex. The level of VEGF in the RPE/choroid was significantly decreased in Aldh1a1–/– mice, and RA-dependent enhancement of VEGF was observed in primary RPE cells. An RA-deficient diet resulted in dorsal choroidal hypoplasia, and simple RA treatment of Aldh1a1–/– pregnant females suppressed choroid hypoplasia in their offspring. We also found downregulation of Sox9 in the dorsal neural retina and RPE of Aldh1a1–/– mice and RPE-specific disruption of Sox9 phenocopied Aldh1a1–/– choroidal development. These results suggest that RAs produced by Aldh1a1 in the neural retina directs dorsal choroidal vascular development via Sox9 upregulation in the dorsal RPE cells to enhance RPE-derived VEGF secretion.

The transcription factor Klf5 is essential for intrahepatic biliary epithelial tissue remodeling after cholestatic liver injury

Under various conditions of liver injury, the intrahepatic biliary epithelium undergoes dynamic tissue expansion and remodeling, a process known as ductular reaction. Mouse models defective in inducing such a tissue-remodeling process are more susceptible to liver injury, suggesting a crucial role of this process in liver regeneration. However, the molecular mechanisms regulating the biliary epithelial cell (BEC) dynamics in the ductular reaction remain largely unclear. Here, we demonstrate that the transcription factor Krüppel-like factor 5 (Klf5) is highly enriched in mouse liver BECs and plays a key role in regulating the ductular reaction, specifically under cholestatic injury conditions. Although mice lacking Klf5 in the entire liver epithelium, including both hepatocytes and BECs (Klf5-LKO (liver epithelial-specific knockout) mice), did not exhibit any apparent phenotype in the hepatobiliary system under normal conditions, they exhibited significant defects in biliary epithelial tissue remodeling upon 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced cholangitis, concomitantly with exacerbated cholestasis and reduced survival rate. In contrast, mice lacking Klf5 solely in hepatocytes did not exhibit any such phenotypes, confirming Klf5s specific role in BECs. RNA-sequencing analyses of BECs isolated from the Klf5-LKO mouse livers revealed that the Klf5 deficiency primarily affected expression of cell cycle-related genes. Moreover, immunostaining analysis with the proliferation marker Ki67 disclosed that the Klf5-LKO mice had significantly reduced BEC proliferation levels upon injury. These results indicate that Klf5 plays a critical role in the ductular reaction and biliary epithelial tissue expansion and remodeling by inducing BEC proliferation and thereby contributing to liver regeneration.

Distinct expression patterns of Flk1 and Flt1 in the coronary vascular system during development and after myocardial infarction

The coronary vascular system is critical for myocardial growth and cardiomyocyte survival. However, the molecular mechanism regulating coronary angiogenesis remains elusive. Vascular endothelial growth factor (VEGF) regulates angiogenesis by binding to the specific receptors Flk1 and Flt1, which results in different functions. Despite the importance of Flk1 and Flt1, their expression in the coronary vasculature remains largely unknown due to the lack of appropriate antibodies for immunostaining. Here, we analyzed multiple reporter mice including Flk1-GFP BAC transgenic (Tg), Flk1-LacZ knock-in, Flt1-DsRed BAC Tg, and Flk1-GFP/Flt1-DsRed double Tg animals to determine expression patterns in mouse hearts during cardiac growth and after myocardial infarction (MI). We found that Flk1 was expressed in endothelial cells (ECs) with a pattern of epicardial-to-endocardial transmural gradients in the neonatal mouse ventricle, which was downregulated in adult coronary vessels with development. In contrast, Flt1 was homogeneously expressed in the ECs of neonatal mouse hearts and expression was maintained until adulthood. After MI, expression of both Flk1 and Flt1 was induced in the regenerating coronary vessels at day 7. Intriguingly, Flk1 expression was downregulated thereafter, whereas Flt1 expression was maintained in the newly formed coronary vessels until 30 days post-MI, recapitulating their expression kinetics during development. This is the first report demonstrating the spatiotemporal expression patterns of Flk1 and Flt1 in the coronary vascular system during development and after MI; thus, this study suggests that these factors have distinct and important functions in coronary angiogenesis.

Fluorescent reporter transgenic mice for in vivo live imaging of angiogenesis and lymphangiogenesis

The study of lymphangiogenesis is an emerging science that has revealed the lymphatic system as a central player in many pathological conditions including cancer metastasis, lymphedema, and organ graft rejection. A thorough understanding of the mechanisms of lymphatic growth will play a key role in the development of therapeutic strategies against these conditions. Despite the known potential of this field, the study of lymphatics has historically lagged behind that of hemangiogenesis. Until recently, significant strides in lymphatic studies were impeded by a lack of lymphatic-specific markers and suitable experimental models compared to those of the more immediately visible blood vasculature. Lymphangiogenesis has also been shown to be a key phenomenon in developmental biological processes, such as cell proliferation, guided migration, differentiation, and cell-to-cell communication, making lymphatic-specific visualization techniques highly desirable and desperately needed. Imaging modalities including immunohistochemistry and in situ hybridization are limited by the need to sacrifice animal models for tissue harvesting at every experimental time point. Moreover, the processes of mounting and staining harvested tissues may introduce artifacts that can confound results. These traditional methods for investigating lymphatic and blood vasculature are associated with several problems including animal variability (e.g., between mice) when replicating lymphatic growth environments and the cost concerns of prolonged, labor-intensive studies, all of which complicate the study of dynamic lymphatic processes. With the discovery of lymphatic-specific markers, researchers have been able to develop several lymphatic and blood vessel-specific, promoter-driven, fluorescent-reporter transgenic mice for visualization of lymphatics in vivo and in vitro. For instance, GFP, mOrange, tdTomato, and other fluorescent proteins can be expressed under control of a lymphatic-specific marker like Prospero-related homeobox 1 (Prox1), which is a highly conserved transcription factor for determining embryonic organogenesis in vertebrates that is implicated in lymphangiogenesis as well as several human cancers. Importantly, Prox1-null mouse embryos develop without lymphatic vessels. In human adults, Prox1 maintains lymphatic endothelial cells and upregulates proteins associated with lymphangiogenesis (e.g., VEGFR-3) and downregulates angiogenesis-associated gene expression (e.g., STAT6). To visualize lymphatic development in the context of angiogenesis, dual fluorescent-transgenic reporters, like Prox1-GFP/Flt1-DsRed mice, have been bred to characterize lymphatic and blood vessels simultaneously in vivo. In this review, we discuss the trends in lymphatic visualization and the potential usage of transgenic breeds in hemangiogenesis and lymphangiogenesis research to understand spatial and temporal correlations between vascular development and pathological progression.