Mohamad Taha

Marked Strain-Specific Differences in the SU5416 Rat Model of Severe Pulmonary Arterial Hypertension

We assessed the pulmonary hemodynamic response to vascular endothelial growth factor receptor, type 2, inhibition using SU5416 (SU) with and without chronic hypoxia (CH) in different background strains and colonies of rats. A single subcutaneous injection of SU (20 mg/kg) or vehicle was administered to different substrains of Sprague-Dawley (SD) rats, and they were compared with Lewis and Fischer rats, with and without exposure to CH (10% O2 for 3 wk). Remarkably, a unique colony of SD rats from Charles River Laboratories, termed the SD-hyperresponsive type, exhibited severe pulmonary arterial hypertension (PAH) with SU alone, characterized by increased right ventricular systolic pressure, right ventricular/left ventricular plus septal weight ratio, and arteriolar occlusive lesions at 7-8 weeks (all P < 0.0001 versus vehicle). In contrast, the other SD substrain from Harlan Laboratories, termed SD-typical type, as well as Fischer rats, developed severe PAH only when exposed to SU and CH, whereas Lewis rats showed only a minimal response. All SD-typical type rats survived for up to 13 weeks after SU/CH, whereas SD-hyperresponsive type rats exhibited mortality after SU and SU/CH (35% and 50%, respectively) at 8 weeks. Fischer rats exposed to SU/CH exhibited the greatest mortality at 8 weeks (78%), beginning as early as 4 weeks after SU and preceded by right ventricle enlargement. Of note, a partial recovery of PAH after 8 weeks was observed in the SD-typical type substrain only. In conclusion, variation in strain, even between colonies of the same strain, has a remarkable influence on the nature and severity of the response to SU, consistent with an important role for genetic modifiers of the PAH phenotype.

Proliferative Versus Degenerative Paradigms in Pulmonary Arterial Hypertension: Have We Put the Cart Before the Horse?

Pulmonary arterial hypertension is believed to be a proliferative disease, triggered by endothelial cell injury and apoptosis and leading to the formation of occlusive vascular lesions caused by growth-dysregulated “cancer-like” cells. However, the current experimental and clinical evidence is not entirely consistent with this paradigm and suggests an alternate interpretation, specifically that microvascular rarefaction may be due to a degenerative process, driven by sustained endothelial cell apoptosis. The “degenerative” paradigm has important implications, not the least of which is that proliferative lesions may be a secondary manifestation of disease rather than the primary cause of microvascular rarefaction, and that regenerative strategies may be needed to restore the microcirculation in established disease . Although there remains much debate about the precise mechanisms of pulmonary arterial hypertension, it is generally accepted that there is functional microvascular rarefaction, resulting in a marked decrease in pulmonary vascular cross-sectional area. Two distinct, and seemingly mutually exclusive, mechanisms have been proposed for the loss of lung microvasculature. The first is a proliferative process that leads to arteriolar occlusion and obliteration, and the second is a degenerative process that leads to drop-out and loss of fragile lung vasculature. Let’s first consider the proliferative hypothesis. The so-called “cancer paradigm” of pulmonary arterial hypertension (PAH) is based on the idea that dysregulated endothelial cell (EC) growth can lead to disordered angiogenesis with the heaping up of intimal cells within the lumen causing arteriolar occlusion and obliteration. Indeed, dysregulated vascular cell growth is integrally linked to the development of plexiform lesions, which represent hallmark features of PAH. This is supported by evidence of changes in biochemical and molecular cellular activity in PAH, similar to those seen in cancer, including a shift in glucose metabolism from mitochondrial glucose oxidation to glycolysis (Warburg effect), activation of cell growth pathways, increased expression of …

Systematic Assessment of Strategies for Lung-targeted Delivery of MicroRNA Mimics

There is considerable interest in the use of synthetic miRNA mimics (or inhibitors) as potential therapeutic agents in pulmonary vascular disease; however, the optimal delivery method to achieve high efficiency, selective lung targeting has not been determined. Here, we sought to investigate the relative merits of different lung-targeted strategies for delivering miRNA mimics in rats. Methods: Tissue levels of a synthetic miRNA mimic, cel-miR-39-3p (0.5 nmol in 50 µL invivofectamine/PBS vehicle) were compared in male rats (n=3 rats/method) after delivery by commonly used lung-targeting strategies including intratracheal liquid instillation (IT-L), intratracheal aerosolization with (IT-AV) or without ventilator assistance (IT-A), intranasal liquid instillation (IN-L) and intranasal aerosolization (IN-A). Intravenous (IV; via jugular vein), intraperitoneal (IP) and subcutaneous (SC) delivery served as controls. Relative levels of cel-miR-39 were quantified by RT-qPCR. Results: At 2 h post delivery, IT-L showed the highest lung mimic level, which was significantly higher than levels achieved by all other methods (from ~10- to 10,000-fold, p<0.05). Mimic levels remained detectable in the lung 24 h after delivery, but were 10- to 100-fold lower. The intrapulmonary distribution of cel-miR-39 was comparable when delivered as either a liquid or aerosol, with evidence of mimic distribution to both the left and right lung lobes and penetration to distal regions. All lung-targeted strategies showed lung-selective mimic uptake, with mimic levels 10- to 100-fold lower in heart and 100- to 10,000-fold lower in liver, kidney and spleen. In contrast, IV, SC and IP routes showed comparable or higher mimic levels in non-pulmonary tissues. Conclusions: miRNA uptake in the lungs differed markedly by up to 4 orders of magnitude, demonstrating that the choice of delivery strategy could have a significant impact on potential therapeutic outcomes in preclinical investigations of miRNA-based drug candidates.

High circulating angiopoietin-2 levels exacerbate pulmonary inflammation but not vascular leak or mortality in endotoxin-induced lung injury in mice

Background Elevated plasma levels of angiopoietin-2 (ANGPT2) have been reported in patients with acute lung injury (ALI); however, it remains unclear whether this increase contributes to, or just marks, the underlying vasculopathic inflammation and leak associated with ALI. Here we investigated the biological consequences of inducing high circulating levels of ANGPT2 in a mouse model of endotoxin-induced ALI. Methods Transgenic mice (ANGPT2OVR) with elevated circulating levels of ANGPT2, achieved through conditional hepatocyte-specific overexpression, were examined from 3 to 72 hours following lipopolysaccharide (LPS)-induced ALI. An aptamer-based inhibitor was used to neutralise the effects of circulating ANGPT2 in LPS-exposed ANGPT2OVR mice. Results Total cells, neutrophils and macrophages, as well as inflammatory cytokines, were significantly higher in bronchoalveolar lavage (BAL) of ANGPT2OVR versus littermate controltTA mice at 48 hours and 6 hours post-LPS, respectively. In contrast, LPS-induced vascular leak, evidenced by total BAL protein levels and lung wet/dry ratio, was unchanged between ANGPT2OVR and controlstTA, while BAL levels of IgM and albumin were decreased in ANGPT2OVR mice between 24 hours and 48 hours suggesting a partial attenuation of vascular leak. There was no significant difference in LPS-induced mortality between ANGPT2OVR and controlstTA. An ANGPT2-neutralising aptamer partially attenuated alveolar cell infiltration while exacerbating vascular leak in LPS-exposed ANGPT2OVR mice, supported by underlying time-dependent changes in the lung transcriptional profiles of multiple genes linked to neutrophil recruitment/adhesion and endothelial integrity. Conclusions Our findings suggest that high circulating ANGPT2 potentiates endotoxin-induced lung inflammation but may also exert other pleiotropic effects to help fine-tune the vascular response to lung injury.

EXPRESS: Systemic Delivery of MicroRNA Mimics with Polyethylenimine Elevates Pulmonary MicroRNA Levels, But Lacks Pulmonary-Selectivity

Reversing pathologic alterations in vascular microRNA (miRNA) expression represents a potential therapeutic strategy for pulmonary hypertension. While polyethylenimine (PEI) has previously been shown to be an effective vehicle for vascular lung-directed delivery of plasmid DNA, it remains unclear whether this utility is generalizable to miRNAs. Here we show that despite elevated lung levels, the intravenous infusion of PEI–miRNA mimic complexes fails to provide lung-selective delivery in rats.