Angiogenesis is a key process in several ocular disorders and cancers. Soluble Flt-1 is an alternatively spliced form of the Flt-1 gene that retains the ligand-binding domain, but lacks the ...membrane-spanning and intracellular kinase domains of the full-length membrane bound Flt-1 (mbFlt-1) protein. Thus, sFlt-1 is an endogenous inhibitor of VEGF-A mediated angiogenesis. Synthetic mopholino oligomers directed against splice site targets can modulate splice variant expression. We hypothesize that morpholino-induced upregulation of sFlt-1 will suppress angiogenesis in clinically relevant models of macular degeneration and breast cancer.
In vivo morpholino constructs were designed to target murine exon/intron 13 junction of the Flt-1 transcript denoted VEGFR1_MOe13; standard nonspecific morpholino was used as control. After nucleofection of endothelial and breast adenocarcinoma cell lines, total RNA was extracted and real-time RT-PCR performed for sFlt-1 and mbFlt-1. Intravitreal injections of VEGFR1_MOe13 or control were done in a model of laser-induced choroidal neovascularization and intratumoral injections were performed in MBA-MD-231 xenografts in nude mice. VEGFR1_MOe13 elevated sFlt-1 mRNA expression and suppressed mbFlt-1 mRNA expression in vitro in multiple cellular backgrounds (p<0.001). VEGFR1_MOe13 also elevated sFlt/mbFlt-1 ratio in vivo after laser choroidal injury 5.5 fold (p<0.001) and suppressed laser-induced CNV by 50% (p = 0.0179). This latter effect was reversed by RNAi of sFlt-1, confirming specificity of morpholino activity through up-regulation of sFlt-1. In the xenograft model, VEGFR1_MOe13 regressed tumor volume by 88.9%, increased sFlt-1 mRNA expression, and reduced vascular density by 50% relative to control morpholino treatment (p<0.05).
Morpholino oligomers targeting the VEGFR1 mRNA exon/intron 13 junction promote production of soluble FLT-1 over membrane bound FLT-1, resulting in suppression of lesional volume in laser induced CNV and breast adenocarcinoma. Thus, morpholino manipulation of alternative splicing offers translational potential for therapy of angiogenic disorders.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Retinopathy of prematurity (ROP), a blinding condition affecting preterm infants, is an interruption of retinal vascular maturation that is incomplete when born preterm. Although ROP demonstrates ...delayed onset following preterm birth, representing a window for therapeutic intervention, there are no curative or preventative measures available for this condition. The in utero environment, including placental function, is increasingly recognized for contributions to preterm infant disease risk. The current study identified a protective association between acute placental inflammation and preterm infant ROP development using logistic regression, with the most significant association found for infants without gestational exposure to maternal preeclampsia and those with earlier preterm birth. Expression analysis of proteins with described ROP risk associations demonstrated significantly decreased placental high temperature requirement A serine peptidase-1 (HTRA-1) and fatty acid binding protein 4 protein expression in infants with acute placental inflammation compared with those without. Within the postnatal peripheral circulation, HTRA-1 and vascular endothelial growth factor-A demonstrated inverse longitudinal trends for infants born in the presence of, compared with absence of, acute placental inflammation. An agnostic approach, including whole transcriptome and differential methylation placental analysis, further identify novel mediators and pathways that may underly protection. Taken together, these data build on emerging literature showing a protective association between acute placental inflammation and ROP development and identify novel mechanisms that may inform postnatal risk associations in preterm infants.
Our understanding of Ewing's sarcoma development mediated by the EWS/FLI fusion protein has been limited by a lack of knowledge regarding the tumor cell of origin. To circumvent this, we analyzed the ...function of EWS/FLI in Ewing's sarcoma itself. By combining retroviral-mediated RNA interference with reexpression studies, we show that ongoing EWS/FLI expression is required for the tumorigenic phenotype of Ewing's sarcoma. We used this system to define the full complement of EWS/FLI-regulated genes in Ewing's sarcoma. Functional analysis revealed that
NKX2.2 is an EWS/FLI-regulated gene that is necessary for oncogenic transformation in this tumor. Thus, we developed a highly validated transcriptional profile for the EWS/FLI fusion protein and identified a critical target gene in Ewing's sarcoma development.
To report the long-term outcomes of transscleral fixation of capsular tension rings (CTR) with intraocular lens (IOL) placement in pediatric patients with ectopia lentis.
John A. Moran Eye Center, ...University of Utah, Salt Lake City, Utah, USA.
Retrospective case series.
Pediatric patients requiring lens extraction with transscleral capsular bag fixation with a CTR and IOL for ectopia lentis between January 2006 and January 2016 were analyzed.
Thirty-seven patients (67 eyes) who had transscleral fixation of the capsular bag using a CTR fixated with 9-0 or 10-0 polypropylene (Prolene), 8-0 polytetrafluoroethylene (Gore-Tex), or 9-0 nylon were identified. The mean age at time of surgery was 7.25 years (2 to 18 years) and the mean follow-up was 35.3 months (0.25 to 120 months). The proportion of eyes showing improvement in corrected distance visual acuity (CDVA) postoperatively was 78.5%, which demonstrated significance with a 95% confidence interval. In the immediate postoperative period, 1 eye developed a hyphema and 1 eye required IOL repositioning. Long-term complications included posterior capsule opacification in 35 eyes (52%) and uveitis–glaucoma–hyphema syndrome in 1 eye (1.5%). Three eyes (4.4 %) required IOL repositioning for spontaneous delayed IOL dislocation, 2 sutured with 8-0 polytetrafluoroethylene at postoperative month 8 and postoperative year 3 and 1 sutured with 9-0 polypropylene at postoperative year 7.
Transscleral fixation of the capsular bag using a CTR improved CDVA and provided IOL stability in pediatric patients with ectopia lentis.
The use of artificial intelligence (AI) and machine learning (ML) in clinical care offers great promise to improve patient health outcomes and reduce health inequity across patient populations. ...However, inherent biases in these applications, and the subsequent potential risk of harm can limit current use. Multi-modal workflows designed to minimize these limitations in the development, implementation, and evaluation of ML systems in real-world settings are needed to improve efficacy while reducing bias and the risk of potential harms. Comprehensive consideration of rapidly evolving AI technologies and the inherent risks of bias, the expanding volume and nature of data sources, and the evolving regulatory landscapes, can contribute meaningfully to the development of AI-enhanced clinical decision making and the reduction in health inequity.
Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, ...with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).
Retinopathy of prematurity (ROP) is a blinding aberrancy of retinal vascular maturation in preterm infants. Despite delayed onset after preterm birth, representing a window for therapeutic ...intervention, we cannot prevent or cure ROP blindness. A natural form of ROP protection exists in the setting of early-onset maternal preeclampsia, though is not well characterized. As ischemia is a central feature in both ROP and preeclampsia, we hypothesized that angiogenesis mediators may underlie this protection. To test our hypothesis we analyzed peripheral blood expression of candidate proteins with suggested roles in preeclamptic and ROP pathophysiology and with a proposed angiogenesis function (HTRA-1, IGF-1, TGFβ-1, and VEGF-A). Analysis in a discovery cohort of 40 maternal-infant pairs found that elevated HTRA-1 (high-temperature requirement-A serine peptidase-1) was significantly associated with increased risk of ROP and the absence of preeclampsia, thus fitting a model of preeclampsia-mediated ROP protection. We validated these findings and further demonstrated a dose-response between systemic infant HTRA-1 expression and risk for ROP development in a larger and more diverse validation cohort consisting of preterm infants recruited from two institutions. Functional analysis in the oxygen-induced retinopathy (OIR) murine model of ROP supported our systemic human findings at the local tissue level, demonstrating that HtrA-1 expression is elevated in both the neurosensory retina and retinal pigment epithelium by RT-PCR in the ROP disease state. Finally, transgenic mice over-expressing HtrA-1 demonstrate greater ROP disease severity in this model. Thus, HTRA-1 may underlie ROP protection in preeclampsia and represent an avenue for disease
, which does not currently exist.
Age-related macular degeneration (AMD) is a progressive neurodegenerative disease that is the world's leading cause of blindness in the aging population. Although the clinical stages and forms of AMD ...have been elucidated, more specific prognostic tools are required to determine when patients with early and intermediate AMD will progress into the advanced stages of AMD. Another challenge in the field has been the appropriate development of therapies for intermediate AMD and advanced atrophic AMD. After numerous negative clinical trials, an anti-C5 agent and anti-C3 agent have recently shown promising results in phase 3 clinical trials, in terms of slowing the growth of geographic atrophy, an advanced form of AMD. Interestingly, both drugs appear to be associated with an increased incidence of wet AMD, another advanced form of the disease, and will require frequent intravitreal injections. Certainly, there remains a need for other therapeutic agents with the potential to prevent progression to advanced stages of the disease. Investigation of the role and clinical utility of non-coding RNAs (ncRNAs) is a major advancement in biology that has only been minimally applied to AMD. In the following review, we discuss the clinical relevance of ncRNAs in AMD as both biomarkers and therapeutic targets.
Retinopathy of prematurity (ROP) is a potentially blinding disorder in premature infants. The underlying pathophysiology is incompletely understood, limiting the prevention and treatment of this ...devastating condition. Current therapies are directed toward management of aberrant neovascularization thought to result from retinal ischemia in the developing preterm retina. The molecular mediators important for development of retinal ischemia and subsequent neovascular pathology are not fully understood. However, oxygen has been shown to be a key mediator of disease and the oxygen environment for preterm infants has been extensively studied. Despite this, the optimal oxygen environment for preterm infants remains unclear and recent works seeking to clarify this relationship demonstrate somewhat disparate findings. These data further substantiate that ROP is a complex disease with multifactorial etiology including genetic and environmental factors. Therefore, while environmental factors such as oxygen are important to our understanding of the disease process and care of preterm infants, identification of the molecular mediators downstream of oxygen which are necessary for development of ROP pathology will be critical to improve prevention, diagnosis and treatment strategies.
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