Koichi Mishima

TGF-β receptor kinase inhibitor enhances growth and integrity of embryonic stem cell–derived endothelial cells

Recent findings have shown that embryonic vascular progenitor cells are capable of differentiating into mural and endothelial cells. However, the molecular mechanisms that regulate their differentiation, proliferation, and endothelial sheet formation remain to be elucidated. Here, we show that members of the transforming growth factor (TGF)-β superfamily play important roles during differentiation of vascular progenitor cells derived from mouse embryonic stem cells (ESCs) and from 8.5–days postcoitum embryos. TGF-β and activin inhibited proliferation and sheet formation of endothelial cells. Interestingly, SB-431542, a synthetic molecule that inhibits the kinases of receptors for TGF-β and activin, facilitated proliferation and sheet formation of ESC-derived endothelial cells. Moreover, SB-431542 up-regulated the expression of claudin-5, an endothelial specific component of tight junctions. These results suggest that endogenous TGF-β/activin signals play important roles in regulating vascular growth and permeability.

Identification of Schlemm's Canal and Its Surrounding Tissues by Anterior Segment Fourier Domain Optical Coherence Tomography

PURPOSE To identify Schlemms canal (SC) and trabecular meshwork (TM) by anterior segment Fourier-domain optical coherence tomography (AS-FD-OCT) with histologic confirmation in enucleated human eyes and to quantitatively evaluate SC and TM in living human eyes. METHODS In enucleated human eyes, the imaging of the anterior chamber angle by AS-FD-OCT was performed before and after surgical expansion of SC with an injection of a viscoelastic material, followed by histologic examination. In 60 living human eyes, the agreement of identification of SC between examiners was evaluated with the Cohens κ values, and the lengths of SC and TM and the area of TM were measured on temporal and nasal sections of the AS-FD-OCT images. RESULTS In enucleated human eyes, SC was observed to be a thin, linear, lucent space in the AS-FD-OCT image obtained with the high-definition raster scan protocol, but not in those obtained with the bi-angle radial scan protocol. This space was enlarged after the SC expansion. In the histologic study, the SC was confirmed to be in the same position as in the AS-FD-OCT images. The κ values of observable SC in living human subjects were 0.92 or higher. The axial length of the SC averaged 347.2 ± 42.3 μm, TM length 466.9 ± 60.7 μm, and TM area 0.0671 ± 0.0058 mm². These measurements showed sufficient repeatability and reproducibility. CONCLUSIONS Using the high-definition images of the AS-FD-OCT, SC and its surrounding tissues were successfully observed in most of the living eyes and were quantitatively evaluated in a noninvasive manner.

Identification of targets of Prox1 during in vitro vascular differentiation from embryonic stem cells: Functional roles of HoxD8 in lymphangiogenesis

During lymphatic development, Prox1 plays central roles in the differentiation of blood vascular endothelial cells (BECs) into lymphatic endothelial cells (LECs), and subsequently in the maturation and maintenance of lymphatic vessels. However, the molecular mechanisms by which Prox1 elicits these functions remain to be elucidated. Here, we identified FoxC2 and angiopoietin-2 (Ang2), which play important roles in the maturation of lymphatic vessels, as novel targets of Prox1 in mouse embryonic-stem-cell-derived endothelial cells (MESECs). Furthermore, we found that expression of HoxD8 was significantly induced by Prox1 in MESECs, a finding confirmed in human umbilical vein endothelial cells (HUVECs) and human dermal LECs (HDLECs). In mouse embryos, HoxD8 expression was significantly higher in LECs than in BECs. In a model of inflammatory lymphangiogenesis, diameters of lymphatic vessels of the diaphragm were increased by adenovirally transduced HoxD8. We also found that HoxD8 induces Ang2 expression in HDLECs and HUVECs. Moreover, we found that HoxD8 induces Prox1 expression in HUVECs and that knockdown of HoxD8 reduces this expression in HDLECs, suggesting that Prox1 expression in LECs is maintained by HoxD8. These findings indicate that transcriptional networks of Prox1 and HoxD8 play important roles in the maturation and maintenance of lymphatic vessels.

Noninvasive observations of peripheral angle in eyes after penetrating keratoplasty using anterior segment fourier-domain optical coherence tomography.

Purpose: To examine iridotrabecular contact (ITC) as a peripheral anterior synechia (PAS) of patients who underwent penetrating keratoplasty (PKP) using anterior segment Fourier-domain optical coherence tomography (OCT). Methods: Retrospective, observational case series. ITC, ITC index, and ITC area of 60 eyes of 52 patients who underwent PKP at the Department of Ophthalmology in the University of Tokyo Hospital (mean follow-up time 102.8 ± 116.1 months postoperation) were calculated using the angle analysis mode of a commercially available anterior segment Fourier-domain OCT system (CASIA; TOMEY, Nagoya, Japan). We analyzed the occurrence of ITC, ITC index, and ITC area with preoperative diagnosis, lens status, operation method, operation frequency, graft size, and intraocular pressure elevation. Results: ITC was observed in 28 (46.7%) of 60 eyes. Average ITC index was 14.0%, and average ITC area was 5.97 mm2. ITC was significantly associated with eyes with bullous keratopathy, infectious keratitis, pseudophakia, PKP with cataract operation, several PKP operations, and large graft size. Conclusions: Anterior segment Fourier-domain OCT is a useful and innovative tool that enables us to observe the anterior chamber angle and examine ITC as PAS of eyes with corneal opacity, even after PKP.

Ultrasound Biomicroscopy in Narrow Peripheral Anterior Chamber Eyes With or Without Peripheral Anterior Synechiae

AimTo compare the configurations of the anterior ocular segment including the anterior chamber (AC) angle and ciliary body between eyes with a narrow AC angle (ACA) with and without peripheral anterior synechiae (PAS). Patients and MethodsOne hundred and one eyes of 101 consecutive patients with a temporal peripheral AC depth one-quarter of the corneal thickness or less were included. Gonioscopy and ultrasound biomicroscopy were performed under light and dark conditions. The existence of PAS was further confirmed with compression gonioscopy with indentation. Eyes with findings suggestive of plateau iris configuration or those with glaucomatous optic neuropathy were carefully excluded. The biometric parameters including the ACA, the angle opening distance at 500 μm, the trabecular-ciliary process distance, the iris-zonule distance, and the scleral-ciliary process angle were determined. ResultsPAS were found in 43 (43%) of the 101 eyes. There were no differences in age, refractive error, or intraocular pressure between PAS-positive and PAS-negative eyes (P>0.1). ACA, iris-zonule distance, scleral-ciliary process angle under light and/or dark conditions were significantly smaller in the PAS-positive eyes than in the PAS-negative eyes (P<0.05). ConclusionsShallow peripheral AC depth and relatively anteriorly located ciliary body was significantly associated with the presence of PAS in eyes with a narrow ACA.

Expression of platelet‐derived growth factor receptor β is maintained by Prox1 in lymphatic endothelial cells and is required for tumor lymphangiogenesis

The lymphatic system plays important roles not only in the physiological processes, such as maintenance of tissue fluid homeostasis, but also in pathological processes including the lymph node metastasis of tumor cells. Therefore, understanding of the molecular mechanisms underlying lymphatic vessel formation is crucial. Previous studies have shown that proliferation and migration of lymphatic endothelial cells (LECs) are activated by multiple types of signals mediated by tyrosine kinase receptors such as vascular endothelial growth factor receptor (VEGFR) 3. Although signals mediated by platelet‐derived growth factor receptor β (PDGFRβ) have been implicated in lymphangiogenesis, the mechanisms explaining how PDGFRβ expression is maintained in LECs remain to be fully elucidated. In the present study, we show that PDGFRβ expression in LECs is maintained by Prox1 transcription factor. Knockdown of Prox1 expression in human dermal LECs decreased the expression of PDGFRβ, leading to the lowered migration of human dermal LECs towards PDGF‐BB. Furthermore, we found that PDGF signals play important roles in inflammatory lymphangiogenesis in a chronic aseptic peritonitis model. Intraperitoneal administration of imatinib, a potent inhibitor of PDGFRβ, and transduction of PDGFRβ/Fc chimeric protein by an adenoviral system both reduced inflammatory lymphangiogenesis induced by thioglycollate in mice. We also found that the expression of PDGFRβ/Fc reduced tumor lymphangiogenesis in a BxPC3 human pancreatic cancer xenograft model. These findings suggest that PDGFRβ is one of the key mediators of lymphatic vessel formation acting downstream of Prox1.

TGF-β Signaling in Embryonic Stem Cell-Derived Endothelial Cells

The roles of the transforming growth factor (TGF)-beta superfamily in vasculogenesis have been implicated by the findings that mutations in genes encoding for various TGF-beta superfamily signaling components exhibit defects in vascular tissues in humans and mice. Embryonic stem cell (ESC)-derived vascular progenitor cells have been shown to differentiate into both endothelial and mural cells. We showed that members of the TGF-beta superfamily play important roles during differentiation of vascular progenitor cells derived from mouse ESC. TGF-beta inhibited proliferation and sheet formation of ESC-derived endothelial cells. Interestingly, SB-431542, a synthetic molecule that inhibits the kinases of receptors for TGF-beta and activin, facilitated proliferation and sheet formation of ESC-derived endothelial cells. We also found that stimulation of ESC-derived endothelial cells with TGF-beta resulted in phosphorylation of both Smad2 and Smadl/5; BMP induced phosphorylation of Smad1/5. In this chapter, we present how to study the cellular and biochemical effects of TGF-beta signals on endothelial cells derived from mouse ESCs.

Factors related to future peripheral anterior synechiae formation on ultrasound biomicroscopy: 7-year follow-up in eyes with shallow peripheral anterior chambers.

Purpose:To study factors associated with future peripheral anterior synechiae (PAS) formation on ultrasound biomicroscopy (UBM) in eyes with a shallow peripheral anterior chamber. Patients and Methods:Fifteen subjects with a shallow peripheral anterior chamber but no PAS in both eyes, in 2001, that had no medical or surgical intervention until 2008 were included in this study. The same examiners performed gonioscopy and UBM at the 3, 6, 9, and 12 o’clock limbal positions under dark and light conditions in 2001 and 2008. A masked examiner performed a quantitative evaluation of the angle configurations in the UBM images. The presence or absence of PAS was determined by compression gonioscopy. The UBM parameters and the number of appositional closures per eye were compared between PAS-positive eyes and PAS-negative eyes. Results:One eye from 15 subjects were randomly selected and included in the analyses. The gonioscopic gradings and UBM parameters did not change significantly (P>0.1) between 2001 and 2008. Seven (47%; 95% confidence interval, 25-70) of 15 eyes developed PAS. In 2001, under dark conditions, the appositional angle closure in 3 to 4 quadrants was found more often (P=0.026) in 7 eyes where PAS developed during the following 7 years than in other 8 eyes. Conclusions:In eyes with a shallow peripheral anterior chamber but no PAS, PAS developed in 47% of eyes followed without intervention for 7 years. PAS was significantly more likely to develop in eyes with the appositional angle closure found in 3 to 4 quadrants under dark conditions.