Effect of an intravitreal antisense oligonucleotide on vision in Leber congenital amaurosis due to a photoreceptor cilium defect.

Photoreceptor ciliopathies constitute the most common molecular mechanism of the childhood blindness Leber congenital amaurosis. Ten patients with Leber congenital amaurosis carrying the c.2991+1655A>G allele in the ciliopathy gene centrosomal protein 290 (CEP290) were treated (ClinicalTrials.gov no. NCT03140969 ) with intravitreal injections of an antisense oligonucleotide to restore correct splicing. There were no serious adverse events, and vision improved at 3 months. The visual acuity of one exceptional responder improved from light perception to 20/400.

Effect of the S-nitrosoglutathione reductase inhibitor N6022 on bronchial hyperreactivity in asthma.

RATIONALE: Patients with asthma demonstrate depletion of the endogenous bronchodilator GSNO and upregulation of GSNOR. OBJECTIVES: An exploratory proof of concept clinical study of N6022 in mild asthma to determine the potential bronchoprotective effects of GSNOR inhibition. Mechanistic studies aimed to provide translational evidence of effect. METHODS: Fourteen mild asthma patients were treated with intravenous N6022 (5 mg) or placebo and observed for 7 days, with repeated assessments of the provocative dose of methacholine causing a 20% fall in FEV1 (methacholine PC20 FEV1), followed by a washout period and crossover treatment and observation. In vitro studies in isolated eosinophils investigated the effect of GSNO and N6022 on apoptosis. MEASUREMENTS AND MAIN RESULTS: This was a negative trial as it failed to reach its primary endpoint, which was change from baseline in methacholine PC20 FEV1 at 24 h. However, our exploratory analysis demonstrated significantly more two dose-doubling increases in PC20 FEV1 for N6022 compared with placebo (21% vs 6%, P < 0.05) over the 7-day observation period. Furthermore, a significant treatment effect was observed in the change in PC20 FEV1 from baseline averaged over the 7-day observation period (mean change: +0.82 mg/ml [N6022] from 1.34 mg/ml [baseline] vs -0.18 mg/ml [placebo] from 1.16 mg/ml [baseline], P = 0.023). N6022 was well tolerated in mild asthmatics. In vitro studies demonstrated enhanced eosinophilic apoptosis with N6022. CONCLUSIONS: In this early phase exploratory proof of concept trial in asthma, N6022 did not significantly alter methacholine PC20 FEV1 at 24 h, but did have a treatment effect at 7 days compared to baseline. Further investigation of the efficacy of S-nitrosoglutathione reductase inhibition in a patient population with eosinophilic asthma is warranted.

Bone morphogenetic protein receptor II regulates pulmonary artery endothelial cell barrier function.

Mutations in bone morphogenetic protein receptor II (BMPR-II) underlie most heritable cases of pulmonary arterial hypertension (PAH). However, less than half the individuals who harbor mutations develop the disease. Interestingly, heterozygous null BMPR-II mice fail to develop PAH unless an additional inflammatory insult is applied, suggesting that BMPR-II plays a fundamental role in dampening inflammatory signals in the pulmonary vasculature. Using static- and flow-based in vitro systems, we demonstrate that BMPR-II maintains the barrier function of the pulmonary artery endothelial monolayer suppressing leukocyte transmigration. Similar findings were also observed in vivo using a murine model with loss of endothelial BMPR-II expression. In vitro, the enhanced transmigration of leukocytes after tumor necrosis factor α or transforming growth factor ß1 stimulation was CXCR2 dependent. Our data define how loss of BMPR-II in the endothelial layer of the pulmonary vasculature could lead to a heightened susceptibility to inflammation by promoting the extravasation of leukocytes into the pulmonary artery wall. We speculate that this may be a key mechanism involved in the initiation of the disease in heritable PAH that results from defects in BMPR-II expression.

Circulating RNA transcripts identify therapeutic response in cystic fibrosis lung disease.

RATIONALE: Circulating leukocyte RNA transcripts are systemic markers of inflammation, which have not been studied in cystic fibrosis (CF) lung disease. Although the standard assessment of pulmonary treatment response is FEV(1), a measure of airflow limitation, the lack of systemic markers to reflect changes in lung inflammation critically limits the testing of proposed therapeutics. OBJECTIVES: We sought to prospectively identify and validate peripheral blood leukocyte genes that could mark resolution of pulmonary infection and inflammation using a model by which RNA transcripts could increase the predictive value of spirometry. METHODS: Peripheral blood mononuclear cells were isolated from 10 patients with CF and acute pulmonary exacerbations before and after therapy. RNA expression profiling revealed that 10 genes significantly changed with treatment when compared with matched non-CF and control subjects with stable CF to establish baseline transcript abundance. Peripheral blood mononuclear cell RNA transcripts were prospectively validated, using real-time polymerase chain reaction amplification, in an independent cohort of acutely ill patients with CF (n = 14). Patients who responded to therapy were analyzed using general estimating equations and multiple logistic regression, such that changes in FEV(1)% predicted were regressed with transcript changes. MEASUREMENTS AND MAIN RESULTS: Three genes, CD64, ADAM9, and CD36, were significant and independent predictors of a therapeutic response beyond that of FEV(1) alone (P < 0.05). In both cohorts, receiver operating characteristic analysis revealed greater accuracy when genes were combined with FEV(1). CONCLUSIONS: Circulating mononuclear cell transcripts characterize a response to the treatment of pulmonary exacerbations. Even in small patient cohorts, changes in gene expression in conjunction with FEV(1) may enhance current outcomes measures for treatment response.

Proof-of-concept trials for lung stem cell therapy.

Proof-of-concept (PoC) trials are an important component of the new therapeutic development paradigm, contributing to the goal of accelerated progression from bench to bedside. Developing a simplified PoC strategy for lung stem or progenitor cell therapy can be helpful in focusing initial efforts and identifying methodologic hurdles in humans at a relatively early stage of development. In this article, the use of lung radiation injury as a model of epithelial injury and regeneration is proposed as a potential PoC strategy. Strengths of the approach include well-understood biology, predictive preclinical and translational models, a tractable human trial design, and the potential to improve outcome in an area of high unmet medical need. While significant hurdles remain, this approach could provide the first interpretable results within 4 years of the decision to proceed, thus greatly accelerating the translation of lung stem cell therapy to humans.

Interaction of interleukin-6 and the BMP pathway in pulmonary smooth muscle.

The majority of familial pulmonary arterial hypertension (PAH) cases are caused by mutations in the type 2 bone morphogenetic protein receptor (BMPR2). However, less than one-half of BMPR2 mutation carriers develop PAH, suggesting that the most important function of BMPR2 mutation is to cause susceptibility to a "second hit." There is substantial evidence from the literature implicating dysregulated inflammation, in particular the cytokine IL-6, in the development of PAH. We thus hypothesized that the BMP pathway regulates IL-6 in pulmonary tissues and conversely that IL-6 regulates the BMP pathway. We tested this in vivo using transgenic mice expressing an inducible dominant negative BMPR2 in smooth muscle, using mice injected with an IL-6-expressing virus, and in vitro using small interfering RNA (siRNA) to BMPR2 in human pulmonary artery smooth muscle cells (PA SMC). Consistent with our hypothesis, we found upregulation of IL-6 in both the transgenic mice and in cultured PA SMC with siRNA to BMPR2; this could be abolished with p38(MAPK) inhibitors. We also found that IL-6 in vivo caused a twofold increase in expression of the BMP signaling target Id1 and caused increased BMP activity in a luciferase-reporter assay in PA SMC. Thus we have shown both in vitro and in vivo a complete negative feedback loop between IL-6 and BMP, suggesting that an important consequence of BMPR2 mutations may be poor regulation of cytokines and thus vulnerability to an inflammatory second hit.

BMP signaling controls PASMC KV channel expression in vitro and in vivo.

Bone morphogenetic proteins (BMPs) have been implicated in the pathogenesis of familial pulmonary arterial hypertension. The type 2 receptor (BMPR2) is required for recognition of all BMPs. Transgenic mice with a smooth muscle cell-targeted mutation in this receptor (SM22-tet-BMPR2(delx4+)) developed increased pulmonary artery pressure, associated with a modest increase in arterial muscularization, after 8 wk of transgene activation (West J, Fagan K, Steudel W, Fouty B, Lane K, Harral J, Hoedt-Miller M, Tada Y, Ozimek J, Tuder R, and Rodman DM. Circ Res 94: 1109-1114, 2004). In the present study, we show that these transgenic mice developed increased right ventricular pressures after only 1 wk of transgene activation, without significant remodeling of the vasculature. We then tested the hypothesis that the increased pulmonary artery pressure due to loss of BMPR2 signaling was mediated by reduced K(V) channel expression. There was decreased expression of K(V)1.1, K(V)1.5, and K(V)4.3 mRNA isolated from whole lung. Western blot confirmed decreased K(V)1.5 protein in these lungs. Human pulmonary artery smooth muscle cells (PASMC) treated with recombinant BMP2 had increased K(V)1.5 protein and macroscopic K(V) current density, which was blocked by anti-K(V)1.5 antibody. In vivo, nifedipine, a selective L-type Ca(2+) channel blocker, reduced RV systolic pressure in these dominant-negative BMPR2 mice to levels seen in control animals. This suggests that activation of L-type Ca(2+) channels caused by reduced K(V)1.5 mediates increased pulmonary artery pressure in these animals. These studies suggest that BMP regulates K(V) channel expression and that loss of this signaling pathway in PASMC through a mutation in BMPR2 is sufficient to cause pulmonary artery vasoconstriction.

Manifestations of cystic fibrosis diagnosed in adulthood.

PURPOSE OF REVIEW: This review highlights the phenotypic features that lead to the diagnosis of cystic fibrosis in adults, and the prognosis of these patients. RECENT FINDINGS: With the widespread availability of genetic testing and a greater appreciation of the clinical spectrum of the disease, the diagnosis of cystic fibrosis is being made with increasing frequency in adults. Clinical features that lead to the diagnosis include respiratory symptoms and chronic airway infection with typical cystic fibrosis pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus, as well as nontuberculous mycobacteria. Often these patients have previously received diagnoses of asthma, chronic bronchitis, or emphysema. Pancreatic insufficiency is much less common in the adult receiving the diagnosis, but pancreatitis occurs with greater frequency. Occasionally, individuals receive diagnoses of apparent single-organ manifestations such as idiopathic pancreatitis or congenital bilateral absence of the vas deferens, but with negligible involvement of the respiratory tract. On rare occasions, patients receiving the diagnosis as adults can present with classic features of the disease. Although lung disease is generally less severe in cystic fibrosis patients receiving the diagnosis as adults than in adult patients who received the diagnosis as infants, the extent of bronchiectasis can nonetheless be severe. The clinical course of patients receiving a diagnosis of cystic fibrosis in adulthood is largely unknown, but frequently they have milder disease and a more favorable prognosis. SUMMARY: Clinicians must be aware of the potential for adults with chronic respiratory tract infections, unexplained bronchiectasis, congenital bilateral absence of the vas deferens, or pancreatitis to have cystic fibrosis despite the age at presentation.

Low-voltage-activated (T-type) calcium channels control proliferation of human pulmonary artery myocytes.

While Ca2+ influx is essential for activation of the cell cycle machinery, the processes that regulate Ca2+ influx in this context have not been fully elucidated. Electrophysiological and molecular studies have identified multiple Ca2+ channel genes expressed in mammalian cells. Ca(v)3.x gene family members, encoding low voltage-activated (LVA) or T-type channels, were first identified in the central nervous system and subsequently in non-neuronal tissue. Reports of a potential role for T-type Ca2+ channels in controlling cell proliferation conflict. The present study tested the hypothesis that T-type Ca2+ channels, encoded by Ca(v)3.x genes, control pulmonary artery smooth muscle cell proliferation and cell cycle progression. Using quantitative RT/PCR, immunocytochemistry, and immunohistochemistry we found that Ca(v)3.1 was the predominant Ca(v)3.x channel expressed in early passage human pulmonary artery smooth muscle cells in vitro and in the media of human pulmonary arteries, in vivo. Selective blockade of Ca(v)3.1 expression with small interfering RNA (siRNA) and pharmacological blockade of T-type channels completely inhibited proliferation in response to 5% serum and prevented cell cycle entry. These studies establish that T-type voltage-operated Ca2+ channels are required for cell cycle progression and proliferation of human PA SMC.

Late diagnosis defines a unique population of long-term survivors of cystic fibrosis.

Although the median survival for patients with cystic fibrosis (CF) is 32.9 years, a small group of patients live much longer. We analyzed the genotype and phenotype of CF patients 40 years and older seen between 1992 and 2004 at the National Jewish Medical and Research Center (n = 55). These patients were divided into two groups according to age at diagnosis: an early diagnosis (ED) group, median age at diagnosis 2.0 years (range 0.1-15 years, n = 28), and a late diagnosis (LD) group, median age of diagnosis 48.8 years (range 24-72.8 years, n = 27). Consistent with the hypothesis that the CFTR genotype affects the age at diagnosis, CFTR DeltaF508 homozygous individuals were more common in the ED group. Although patients in the ED group were predominantly male, the majority of LD patients were female. Patients with CF diagnosed late had a significantly lower prevalence of pancreatic insufficiency and CF-related diabetes, and better lung function. Fewer patients in the LD groups were infected with Pseudomonas aeruginosa, whereas a greater percentage had cultures positive for nontuberculous mycobacteria. This is the largest cohort of older patients with CF described to date, and our findings indicate that patients diagnosed as adults differ distinctly from survivors of long-term CF diagnosed as children.

Tumor necrosis factor-related apoptosis-inducing ligand can induce apoptosis in subsets of premalignant cells.

During the transformation from a normal to a malignant cell, several mutations are required to bypass the pathways responsible for controlling proliferation. Premalignant cells have acquired some, but not all of these mutations and consequently have not yet attained a malignant phenotype characterized by tumor formation in vivo. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in malignant cells while sparing normal ones and is currently being considered as adjuvant therapy for various human malignancies. Whether TRAIL is effective in inducing apoptosis in premalignant cells is unclear, however. We studied the effect of TRAIL on two human premalignant cell lines the SV7tert and HA1E cells. Both cell lines had been immortalized by the addition of simian virus 40 large T antigen and the telomerase subunit hTERT, but had not been transformed into malignant cells. TRAIL initiated apoptosis by activating both the mitochondrial-independent and -dependent apoptotic pathways in both cell lines at relatively low doses whereas it had no effect on normal human pulmonary artery smooth muscle cells even at high doses. These results suggest that TRAIL can induce apoptosis in premalignant cells and suggests a novel therapy for the treatment of premalignant lesions in vivo.

Cardioprotective and vasomotor effects of HO activity during acute and chronic hypoxia.

Prolonged hypoxia leads to the development of pulmonary hypertension. Recent reports have suggested enhancement of heme oxygenase (HO), the major source of intracellular carbon monoxide (CO), prevents hypoxia-induced pulmonary hypertension and vascular remodeling in rats. Therefore, we hypothesized that inhibition of HO activity by tin protoporphyrin (SnPP) would exacerbate the development of pulmonary hypertension. Rats were injected weekly with either saline or SnPP (50 micromol/kg) and exposed to hypobaric hypoxia or room air for 5 wk. Pulmonary and carotid arteries were catheterized, and animals were allowed to recover for 48 h. Pulmonary and systemic pressures, along with cardiac output, were recorded during room air and acute 10% O2 breathing in conscious rats. No difference was detected in pulmonary artery pressure between saline- and SnPP-treated animals in either normoxic or hypoxic groups. However, blockade of HO activity altered both systemic and pulmonary vasoreactivity to acute hypoxic challenge. Despite no change in baseline pulmonary artery pressure, all rats treated with SnPP had decreased ratio of right ventricular (RV) weight to left ventricular (LV) plus septal (S) weight (RV/LV + S) compared with saline-treated animals. Echocardiograms suggested dilatation of the RV and decreased RV function in hypoxic SnPP-treated rats. Together these data suggest that inhibition of HO activity and CO production does not exacerbate pulmonary hypertension, but rather that HO and CO may be involved in mediating pulmonary and systemic vasoreactivity to acute hypoxia and hypoxia-induced RV function.

Pulmonary hypertension in transgenic mice expressing a dominant-negative BMPRII gene in smooth muscle.

Bone morphogenetic peptides (BMPs), a family of cytokines critical to normal development, were recently implicated in the pathogenesis of familial pulmonary arterial hypertension. The type-II receptor (BMPRII) is required for recognition of all BMPs, and targeted deletion of BMPRII in mice results in fetal lethality before gastrulation. To overcome this limitation and study the role of BMP signaling in postnatal vascular disease, we constructed a smooth muscle-specific transgenic mouse expressing a dominant-negative BMPRII under control of the tetracycline gene switch (SM22-tet-BMPRII(delx4+) mice). When the mutation was activated after birth, mice developed increased pulmonary artery pressure, RV/LV+S ratio, and pulmonary arterial muscularization with no increase in systemic arterial pressure. Studies with SM22-tet-BMPRII(delx4+) mice support the hypothesis that loss of BMPRII signaling in smooth muscle is sufficient to produce the pulmonary hypertensive phenotype.

Second-strand genome conversion of adeno-associated virus type 2 (AAV-2) and AAV-5 is not rate limiting following apical infection of polarized human airway epithelia.

Recombinant adeno-associated virus type 5 (rAAV-5) is known to efficiently transduce airway epithelia via apical infection. In contrast, rAAV-2 has been shown to be inherently ineffective at transducing airway epithelia from the apical surface. However, tripeptide proteasome inhibitors (such as LLnL) can dramatically enhance rAAV-2 transduction from the apical surface of human polarized airway epithelia by modulating the intracellular trafficking and processing of the virus. To further investigate potential differences between rAAV-2 and rAAV-5 that might explain their altered ability to transduce airway epithelia from the apical membrane, we examined the functional involvement of the ubiquitin/proteasome pathway and rate-limiting aspects of second-strand synthesis for these two rAAV serotypes. To this end, we conducted studies to compare the extent to which LLnL alters transduction efficiencies with both rAAV-2 and rAAV-2/5 by using luciferase and enhanced green fluorescent protein (EGFP) reporter vectors. Our results demonstrate that the coadministration of LLnL at the time of viral infection significantly enhanced transduction of both rAAV-2/5 and rAAV-2 from the apical surface of airway epithelia. Although rAAV-2/5 was slightly more effective at transducing epithelia from the apical membrane, rAAV-2 transduction was superior to that of rAAV-2/5 in the presence of proteasome inhibitors. Interestingly, the basolateral membrane entry pathways for both serotypes were not significantly affected by the addition of LLnL, which suggests that apical and basolateral infectious pathways possess distinctive intracellular processing pathways for both rAAV-2 and rAAV-5. Studies comparing the transduction of short self-complementary (scAAV) to full-length conventional AAV EGFP vectors suggested that second-strand synthesis of rAAV genomes was not rate limiting for either serotype or altered by proteasome inhibitors following apical infection of polarized airway epithelia. These findings suggest that both rAAV-2 and rAAV-5 share similar intracellular viral processing barriers that involve the ubiquitin/proteasome system, but do not appear to involve second-strand synthesis.

Mevastatin can cause G1 arrest and induce apoptosis in pulmonary artery smooth muscle cells through a p27Kip1-independent pathway.

Advanced pulmonary arterial hypertension is characterized by extensive vascular remodeling that is usually resistant to vasodilator therapy. Mevastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting step for cholesterol synthesis. HMG-CoA reductase inhibitors have been shown to upregulate the cyclin-dependent kinase inhibitor p27Kip1 and to block cell proliferation through cholesterol-independent pathways. The aim of this study was to determine the effect of mevastatin on DNA synthesis, cell cycle progression, and cell proliferation in rat pulmonary artery smooth muscle cells (PASMCs). We found that mevastatin induced G1 arrest and decreased DNA synthesis in rat PASMCs and did so in association with an increase in both total and cyclin E-bound p27Kip1. This caused a marked decrease in cyclin E kinase activity, which suggests an important role for p27Kip1 in the ability of mevastatin to induce G1 arrest. However, in PASMCs lacking functional p27Kip1, mevastatin still decreased cyclin E kinase activity, caused G1 arrest, and decreased DNA synthesis. In p27Kip1-deficient PASMCs, mevastatin induced a greater reduction of cyclin E protein levels (to 35% of control) than in wild-type cells (to 70% of control) and also reduced the phosphorylation of cdk2 on threonine 160. Mevastatin also caused apoptosis in both wild-type and p27Kip1-deficient PASMCs and was able to do so at a dose that did not induce cell cycle arrest. These data suggest that HMG-CoA reductase inhibitors can both inhibit cell proliferation and induce apoptosis in PASMCs through p27Kip1-independent pathways and may be important therapeutic agents in pulmonary arterial hypertension.

Nitric oxide and protein nitration in the cystic fibrosis airway.

Cystic fibrosis (CF), characterized by chronic airway infection and inflammation, ultimately leads to respiratory failure. Exhaled nitric oxide (NO), elevated in most inflammatory airway diseases, is decreased in CF, suggesting either decreased production or accelerated metabolism of NO. The present studies performed on two groups of CF patients provide further support for a disordered NO airway metabolism in CF respiratory tract disease. Despite confirmation of subnormal NOS2 in the CF airway epithelium, alternative isoforms NOS1 and NOS3 were present, and inflammatory cells in the CF airway expressed abundant NOS2. Increased immunohistochemical staining for nitrotyrosine was demonstrated in lung tissues from patients with CF as compared to control. To our knowledge, this is the first report localizing nitrotyrosine in diseased CF lung tissue. While the relative NOS2 deficiency in CF respiratory tract epithelium may contribute to the lower expired NO levels, these results suggest that increased metabolism of NO is also present in advanced CF lung disease. The significance of altered NO metabolism and protein nitration in CF remains to be fully elucidated.