Morpholino-Mediated Increase in Soluble Flt-1 Expression Results in Decreased Ocular and Tumor Neovascularization

BACKGROUND: 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. METHODS AND FINDINGS: 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). CONCLUSIONS: 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

Intra-tumoral VEGFR1_MOe13 injection results in regression of established MBA-MD-231 human breast adenocarcinoma xenograft tumors and decreased tumor vascularity.

<p>a. MB-MDA-231 breast cancer cells were grown as xenografts in female nude mice for 2 weeks prior to beginning treatment with either a standard morpholino or VEGFR1_MOe13 designed to target the murine VEGFR1 transcript. Morpholino treated tumors demonstrate size regression after a 4 week treatment course as demonstrated by volume change analysis of 5 individual tumors within each treatment condition. p = 0.04 b–c. Following treatment course, RNA was extracted from xenograft tumors and soluble or membrane bound Flt1 levels were measured using real-time RT-PCR. Error bars indicate variation between individual PCR reactions per tumor sample. d. Following the treatment course, VEGFR1_MOe13 or standard morpholino xenograft tumors were sectioned and stained with isolectin as a measure of vascularity. Individual bars represent sections analyzed thoughout each tumor sample.</p

VEGFR1_MOe13 increases sFLT-1 and decreases mbFLT-1 mRNA in MCF-7 and MBA-MD-231 breast adenocarcinoma cell lines.

<p>MCF7 or MBA-MD-231 human breast adenocarcinoma cells were electroporated with VEGFR1_MOe13 and (a; c) sFLT-1 and (b; d) mbFLT-1 mRNA levels assessed at 72 hours using real time PCR (n = 3). Data were normalized to GAPDH mRNA levels and normal MCF7 or MBA-MD-231 cells were used a 1.0. *p<0.01.</p

VEGFR1_MOe13 localizes to the nucleus and increases sFLT-1 expression in human endothelial vein cells (HUVEC).

<p>(a) Fluorescently tagged VEGFR1_MOe13 (F-MO) or standard morpholino (std-MO) were electroporated into HUVECs. After 48 hours fluorescence was assessed using light microscopy. Colocalization with DAPI staining represents nuclear localization of morpholino constructs. HUVECs were electroporated with VEGFR1_MOe13, VEGFR1_MOi13, a combination of VEGFR1_MOe13 and VEGFR1_MOi13, Standard_MO. All morpholino sequences were designed to target the human VEGFR1 transcript. (b) mbFLT-1 mRNA (n = 6) or (c) sFLT-1 mRNA expression (n = 6) were assessed using real time PCR. Values were normalized to GAPDH mRNA and normal HUVEC was used as 1.0. (d) sFLT protein expression in culture medium was determined by ELISA (n = 3). Data shows sFLT protein at 96 h – 48 h. Error bar is S.E.M. Each p-value was calculated by two-tail student's t-test against normal HUVEC.</p

VEGFR1_MOe13 inhibits laser-induced CNV in vivo.

<p>a. sFlt/mFlt mRNA ratio in the retina treated with PBS, Standard_MO and VEGFR1_MOe13 (n = 4). Representative images of laser CNV injected with b PBS, c Standard_MO and d VEGFR1_MOe13 designed to target the murine VEGFR1 transcript. e The averages of laser CNV volumes (n = 11–14). Error bar is S.E.M. p-values were calculated by two-tail student's t test.</p

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used