Hyaluronidase-1-mediated glycocalyx impairment underlies endothelial abnormalities in polypoidal choroidal vasculopathy.

BACKGROUND: Polypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is a global leading cause of vision loss in older populations. Distinct from typical AMD, PCV is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis. RESULTS: We cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity, as demonstrated by a significant 1.5-fold upregulation of Krüppel-like factor 2 (KLF2) expression, a flow-responsive transcription factor. Activation of KLF2 might in turn modulate PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples. CONCLUSIONS: Hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.

Segmentation and characterization of interscapular brown adipose tissue in rats by multi-parametric magnetic resonance imaging.

OBJECTIVE: The aim was to auto-segment and characterize brown adipose, white adipose and muscle tissues in rats by multi-parametric magnetic resonance imaging with validation by histology and UCP1. MATERIALS AND METHODS: Male Wistar rats were randomized into two groups for thermoneutral (n = 8) and cold exposure (n = 8) interventions, and quantitative MRI was performed longitudinally at 7 and 11 weeks. Prior to imaging, rats were maintained at either thermoneutral body temperature (36 ± 0.5 °C), or short term cold exposure (26 ± 0.5 °C). Neural network based automatic segmentation was performed on multi-parametric images including fat fraction, T2 and T2* maps. Isolated tissues were subjected to histology and UCP1 analysis. RESULTS: Multi-parametric approach showed precise delineation of the interscapular brown adipose tissue (iBAT), white adipose tissue (WAT) and muscle regions. Neural network based segmentation results were compared with manually drawn regions of interest, and showed 96.6 and 97.1% accuracy for WAT and BAT respectively. Longitudinal assessment of the iBAT volumes showed a reduction at 11 weeks of age compared to 7 weeks. The cold exposed group showed increased iBAT volume compared to thermoneutral group at both 7 and 11 weeks. Histology and UCP1 expression analysis supported our imaging results. CONCLUSION: Multi-parametric MR based neural network auto-segmentation provides accurate separation of BAT, WAT and muscle tissues in the interscapular region. The cold exposure improves the classification and quantification of heterogeneous BAT.

MiR-135b is a direct PAX6 target and specifies human neuroectoderm by inhibiting TGF-ß/BMP signaling.

Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development, and recently, the TF PAX6 was shown to be critical for human NE specification. However, microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach, we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs, including hsa-miR-135b. MiR-135b was activated during NE development, and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-ß and BMP signaling pathways, thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage.

Histological evaluation of osteogenesis of 3D-printed poly-lactic-co-glycolic acid (PLGA) scaffolds in a rabbit model.

Utilizing a suitable combination of lactide and glycolide in a copolymer would optimize the degradation rate of a scaffold upon implantation in situ. Moreover, 3D printing technology enables customizing the shape of the scaffold to biometric data from CT and MRI scans. A previous in vitro study has shown that novel 3D-printed poly-lactic-co-glycolic acid (PLGA) scaffolds had good biocompatibility and mechanical properties comparable with human cancellous bone, while they could support proliferation and osteogenic differentiation of osteoblasts. Based on the previous study, this study evaluated PLGA scaffolds for bone regeneration within a rabbit model. The scaffolds were implanted at two sites on the same animal, within the periosteum and within bi-cortical bone defects on the iliac crest. Subsequently, the efficacy of bone regeneration within the implanted scaffolds was evaluated at 4, 12 and 24 weeks post-surgery through histological analysis. In both the intra-periosteum and iliac bone defect models, the implanted scaffolds facilitated new bone tissue formation and maturation over the time course of 24 weeks, even though there was initially observed to be little tissue ingrowth within the scaffolds at 4 weeks post-surgery. Hence, the 3D-printed porous PLGA scaffolds investigated in this study displayed good biocompatibility and are osteoconductive in both the intra-periosteum and iliac bone defect models.

Proliferation and differentiation of human osteoblasts within 3D printed poly-lactic-co-glycolic acid scaffolds.

Bone repair and regeneration can be enhanced through implantation of biocompatible and biodegradable scaffolds, which serve primarily as osteoconductive moieties. In this study, the mechanical properties and microenviroment of 3D printed poly-lactic-co-glycolic acid (PLGA) scaffolds are examined. Additionally, the proliferation and differentiation of human fetal osteoblasts are evaluated after 3 weeks of in vitro culture on the scaffolds. The results showed that the PLGA scaffolds examined had mechanical properties similar to that of trabecular bone, but was still much weaker compared to cortical bone. In addition to general porosity, the PLGA scaffolds also had micropores within macropore walls. Cultured human osteoblasts could proliferate upon seeding on the PLGA scaffolds. Alkaline phosphatase activity and osteonectin expression of the osteoblasts cultured on the PLGA scaffolds remained stable over three weeks, whilst expression of collagen type I and osteopontin decreased. The alkaline phosphatase activity of osteoblasts cultured on PLGA scaffolds is comparable with that from two commercially-available scaffolds - OPLA and collagen scaffolds (Becton-Dickinson (BD) Inc., Franklin Lakes, NJ, USA). Hence, the results suggested that the PLGA scaffolds examined are conducive for promoting osteogenesis.