Ann M. Reynolds

Targeted gene delivery of BMPR2 attenuates pulmonary hypertension

Pulmonary arterial hypertension (PAH) remains a fatal disease despite modern pharmacotherapy. Mutations in the gene for bone morphogenetic protein receptor type II (BMPR2) lead to reduced BMPR2 expression, which is causally linked to PAH. BMPR2 is predominantly expressed on pulmonary endothelium and has complex interactions with transforming growth factor (TGF)-&bgr; signalling mechanisms. Our objectives were to assess the effect on PAH of upregulating BMPR2 by targeted adenoviral BMPR2 gene delivery to the pulmonary vascular endothelium. We used two established rat models of PAH: chronic hypoxia and monocrotaline (MCT). In both hypertensive models, those receiving BMPR2 had less right ventricular hypertrophy, less pulmonary vascular resistance, improved cardiac function and reduced vascular remodelling. In the MCT model, there was an increase in TGF-&bgr;, which was prevented by BMPR2 treatment. In vitro, TGF-&bgr;1-induced endothelial–mesenchymal transition (EndMT) in human pulmonary microvascular endothelial cells, which was associated with reduced BMPR2 expression. EndMT was partially ameliorated by stimulating BMPR2 signalling with appropriate ligands even in the ongoing presence of TGF-&bgr;1. Collectively, these results indicate therapeutic potential for upregulation of the BMPR2 axis in PAH, which may be, in part, mediated by countering the remodelling effects of TGF-&bgr;.

Cytokines enhance airway smooth muscle contractility in response to acetylcholine and neurokinin A

In vivo airway hyperresponsiveness has been demonstrated following inhalation of specific cytokines in normal individuals. Whether this airway hyperresponsiveness results from a direct effect of cytokines on airway smooth muscle contractility, or via changes in airway wall structure is not known. The aim of the present study was to determine the effect of the pro‐inflammatory cytokines interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) on airway smooth muscle contractility in vitro.

BMPR2 gene therapy for PAH acts via Smad and non-Smad signalling.

Pulmonary arterial hypertension (PAH) continues to be a fatal disease and is associated with downregulation of bone morphogenetic protein receptor type‐2 (BMPR2). Our approach is to upregulate BMPR2 in the pulmonary vasculature allowing us to examine the changes in endothelial cell signalling and better understand what pathways are altered when disease is attenuated using this treatment approach.

Interleukin-1beta and tumour necrosis factor-alpha increase microvascular leakage in the guinea pig trachea.

Objective: Airway microvascular leakage is considered to be an important component of airway inflammation in asthma. In the present study we examined the effect of interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNFα) on airway microvascular leakage in vivo.

Tachykinin-induced bronchoconstriction in sheep is NK-1 receptor mediated and exhibits tachyphylaxis

Objective: Tachykinins are mediators of airway hyper‐reactivity and inflammation. There is in vitro evidence that ovine responses to tachykinins correlate closely to human responses. This study was designed to characterize the effect of intravenously administered tachykinins on sheep lung resistance in vivo to determine the effect of dose timing on reproducibility of responses and the induction of tachyphylaxis. We then used this information to help further characterize the response with several pharmacological agents.

Tachykinins contribute to the acute airways response to allergen in sheep actively sensitized to Ascaris suum

Abstract Tachykinins, found in sensory nerves, have effects in the airways which suggest that they may contribute to the pathogenesis of asthma. We aimed to find evidence for tachykinin involvement in the immediate airway response to allergen in a sheep model of experimental asthma. Twenty‐four sheep were actively sensitized to Ascaris suum, then challenged with nebulized Ascaris extract in a dose‐response fashion. Change in lung resistance (RL) in response to challenge was measured. Responder sheep (those with an increase in RL of >100% over baseline) that had reproducible responses over three challenges were identified (n= 4 sheep) and a PC100 (number of breaths of extract required to induce a 100% increase in RL) was determined. The effect of the neutral endopeptidase inhibitor phosphoramidon, the NK‐1 receptor‐specific antagonist CP 96, 345 and capsaicin desensitization on the RL response to Ascaris challenge was then assessed. Administration of phosphoramidon before Ascaris decreased the PC100 to 31 ± 7% of the PC100 seen with Ascaris alone (P<0.05), whereas CP 96,345 and capsaicin desensitization increased the PC100 to 285 ± 41% and 555 ± 93% respectively (P<0.05 for both). These findings suggest that endogenous tachykinins are released in response to allergen challenge and that they contribute to the immediate increase in RL.

Tachykinin NK2 receptors predominantly mediate tachykinin-induced contractions in ovine trachea

In vitro studies were conducted to characterize the contractile effects of tachykinins in normal ovine trachea with a view in the future to compare tachykinin contractile responses in allergic tissue. Tracheal smooth muscle strips were prepared for in vitro studies of isometric contraction in response to cumulative addition of carbachol, acetylcholine, histamine, neuropeptide gamma, substance P, neurokinin A, neurokinin B, [Sar9, Met(O2)11]substance P, [Nle10]neurokinin A-(4-10), and [Succinyl-Asp6, Me-Phe8]substance P-(6-11) (senktide). The rank order of potency was neuropeptide gamma > carbachol > neurokinin A > or = [Nle10]neurokinin A-(4-10) > acetylcholine > or = histamine. Phosphoramidon enhanced the contractile response to neurokinin A and substance P, but not to neuropeptide gamma, [Sar9, Met(O2)11]substance P or senktide. Repeated cumulative concentration responses for acetylcholine, substance P, neurokinin A, [Sar9, Met(O2)11]substance P and histamine were also conducted to test for tachyphylaxis. No tachyphylaxis to acetylcholine, substance P, or neurokinin A was observed, however, [Sar9, Met(O2)11]substance P and histamine did exhibit tachyphylaxis. Atropine had no effect on tracheal contractions to neurokinin A and substance P, while [Sar9, Met(O2)11]substance P contractions were atropine sensitive. Pyrilamine did not affect substance P-induced tracheal smooth muscle contractions, indicating that the response to substance P was not mediated by histamine release. These results show that, in vitro, natural tachykinins induce tracheal smooth muscle contraction predominantly by a direct effect mediated by tachykinin NK2 receptors, and a small tachykinin NK1 receptor mediated cholinergic mechanism.

Corrigendum to “In vitro susceptibility to the pro-apoptotic effects of TIMP-3 gene delivery translates to greater in vivo efficacy versus gene delivery for TIMPs-1 or -2” [Lung Cancer 53 (3) (September 2006) 273–284]

Please cite this article in press as: K.M. Finan, et al., Corrigendum to “In vitro susceptibility to the pro-apoptotic effects of TIMP-3 gene delivery translates to greater in vivo efficacy versus gene delivery for TIMPs-1 or -2” [Lung Cancer 53 (3) (September 2006) 273–284], Lung Cancer (2016), http://dx.doi.org/10.1016/j.lungcan.2016.06.002 Fig. 8 The authors would like to apologise for any inconvenience caused.

811. ACE-Targeted eNOS and BMPR2 Gene Therapy Attenuates Pulmonary Hypertension in a Chronic Hypoxia Rat Model

Idiopathic pulmonary arterial hypertension is a fatal disease characterized by vascular remodeling and right ventricular failure. Modern vasodilator therapies improve prognosis but ultimately most patients succumb or require heart-lung transplantation. We previously developed targeted adenoviral (Ad) strategies for pulmonary vascular gene delivery based on the use of a bispecific conjugate (Fab-9B9) that targets Ad infection to angiotensin converting enzyme (ACE) expressed on pulmonary vascular endothelium. In the current study we determined whether targeting achieved therapeutic gains when using Ad gene delivery of endothelial nitric oxide synthase (eNOS), then evaluated a more novel approach using targeted delivery of the gene for bone morphogenetic protein receptor type 2, based on the knowledge that mutations in this receptor and defects in BMPR2 signaling account for many of the inherited forms of IPAH. Rats (n=6) were injected with 5|[times]|109 pfu of either irrelevant virus, AdCMVeNOS, AdCMVeNOS+Fab9B9 or saline (PBS), then exposed to 10% oxygen atmosphere for three weeks, then right ventricular systolic pressure (RVSP), mean pulmonary artery pressure (PAP) and right ventricular hypertrophy (Fulton Index, FI) were determined. A concurrent group of rats maintained in normoxic conditions was used as a control. Hypoxia lead to increased RVSP (42.2|[plusmn]|4.6mmHg), PAP (34.5|[plusmn]|2.8 mmHg) and FI (0.48|[plusmn]|0.02) vs normoxic controls (22.3|[plusmn]|0.8, 19.4|[plusmn]|1.2 and 0.26|[plusmn]|0.01 respectively). AdCMVeNOS attenuated RVSP(33.9|[plusmn]|1.9), PAP(27|[plusmn]|2.3) and FI(0.41|[plusmn]|.03), with a further therapeutic gain seen with Fab-9B9 targeting (RVSP 28.0|[plusmn]|1.8). A separate experiment using irrelevant virus+Fab-9B9, AdCMVBMPR2+Fab-9B9 or PBS showed that BMPR2 gene delivery attenuated the hypoxia-induced increase in RVSP, PAP and FI by 27%, 34% and 50% respectively. Irrelevant Ad alone or with Fab-9B9 had no effect. Thus, the novel findings are that Ad-mediated eNOS delivery attenuates chronic hypoxia-induced PHT, and this effect is improved with targeting. This is also the first demonstration that upregulation of BMPR2 signaling attenuates pulmonary hypertension, suggesting a therapeutic strategy for patients with BMPR2 mutations.

952. Delivery of Tissue Inhibitor of Metalloproteinase 3 Inhibits Growth of Lung Cancer Tumours In Vivo

Lung cancer causes more cancer deaths than cancers related to colon, breast and prostate combined. More primary lung cancers are now diagnosed in former smokers than current smokers and this trend is set to continue. Despite advances in chemotherapeutic agents current treatment strategies are not effective enough in reducing the burden of disease, producing survival benefit or improving quality of life. There is no doubt that new treatment strategies are necessary to improve the outlook from this disease. Gene therapy exploits our expanding knowledge of the molecular basis of cancer using a number of cancer modifying techniques. The extracellular matrix (ECM) provides the scaffolding for all cells and is the environment in which all tissues and cells exist. In order for tissues to change their form the ECM must be degraded and the tissue remodelled. Matrix Metalloproteinases (MMPs) are the proteins that regulate ECM functions. Through a variety of complex mechanisms they control cell differentiation, proliferation and migration. The Tissue inhibitors of Metalloproteinases (TIMPs) are a family of multifunctional proteins named for their ability to inhibit MMPs. TIMP 3 also has the unique ability to directly induce apoptosis in certain tumour types. Hypothesis: In vitro delivery of AdCMVTIMP-3 induces apoptosis and cell death in lung cancer cells, that AdCMVTIMP-3 has a significant bystander effect and in vivo delivery of AdCMVTIMP-3 to implanted subcutaneous A549 tumours reduces tumour growth. Aims: To demonstrate the in vitro and in vivo apoptotic and cell killing effects of AdCMVTIMP-3. Methods: Lung cancer cells A549 were plated at known concentrations and then infected with adenoviral vectors carrying the genes for TIMP-1, -2 and 3. Changes consistent with apoptosis were observed at 48-66 hours post infection. A variety of flow cytometric analyses including Annexin V, Propidium Iodide, 7AAD and Single Stranded DNA confirm that AdCMVTIMP-3 induces apoptosis and cell death. Elevated Caspace, p53, BAX/Bcl2 and Fas levels show activation of the caspase cascade that regulates programmed cell death. MTT assays confirm reductions in cell numbers and demonstrate the significant bystander effects of AdCMVTIMP-3. In vivo analysis of A549 tumours implanted subcutaneously in nude mice shows delivery of AdCMVTIMP-3 inhibits tumour growth. TUNEL analysis of paraffin embedded tissue sections of these tumours shows evidence of increased numbers of apoptotic cells when compared to uninjected controls and those injected with control virus. Conclusion: Delivery of AdCMVTIMP-3 induces apoptosis and cell death in lung cancer cells and this can be demonstrated in vitro and in vivo.