Search Results (18)

Background: Human Immunodeficiency Virus (HIV) infections remain a source of cardiopulmonary complications among people receiving antiretroviral therapy. Still to this day, pulmonary hypertension (PH) severely affects the prognosis in this patient population. The persistent expression of HIV proteins, even during viral suppression, has been implicated in vascular dysfunction; however, little is known about the specific effects of these proteins on the pulmonary vasculature. This study investigates the impact of Nef variants derived from HIV-positive pulmonary hypertensive and normotensive donors on pulmonary vascular cells in vitro. Methods: We utilized well-characterized Nef molecular constructs to examine their effects on cell adhesion molecule gene expression (ICAM1, VCAM1, and SELE), pro-apoptotic gene expression (BAX, BAK), and vasoconstrictive endothelin-1 (EDN1) gene expression in endothelial nitric oxide synthase (eNOS) nitric oxide and the production and secretion of pro-inflammatory cytokines over 24, 48, and 72 h post-transfections with Nef variants. Results: HIV Nef variants SF2, NA7, and PH-associated Fr17 and 3236 induced a significant increase in adhesion molecule gene expression of ICAM1, VCAM1, and SELE. Pulmonary normotensive Nef 1138 decreased ICAM1 gene expression, but had increased VCAM1. PH Nef ItVR showed a consistent decrease in ICAM1 and no changes in SELE and VCAM1 expression. Further gene expression analyses of pro-apoptotic genes BAX and BAK demonstrated that Nef NA7, SF2, normotensive Nef 1138, and PH Nef Fr8, Fr9, Fr17, and 3236 variants significantly increased gene expression for apoptosis. Normotensive Nef 1138, as well as PH Nef Fr9 and ItVR, all displayed a statistically significant decrease in BAX expression. The expression of EDN1 had a statistically significant increase in samples treated with Nef NA7, SF2, normotensive Nef 2044 and PH Nef 3236, Fr17, and Fr8. Notably, PH-associated Nef variants sustained pro-inflammatory cytokine production, including IL-2, IL-4, and TNFα, while anti-inflammatory cytokine levels remained insufficient. Furthermore, eNOS was transiently upregulated by all Nef variants except for normotensive Nef 2044. Conclusions: The distinct effects of Nef variants on pulmonary vascular cell biology highlight the complex interplay between Nef, host factors, and vascular pathogenesis according to the variants. Full article

Linear DNA constructs are used in gene delivery and therapy application due to their capacity of integration into the mammalian genome, offering stable transgene expression. Compared to circular plasmids, linear DNA also has the advantage that its dimension and steric hindrance are directly correlated to the length of the nucleotide chain. These considerations make linear DNA an effective choice for gene delivery pilot studies, where formulations and transfection efficiency calculations are studied considering the nucleic acid dimensions. Meanwhile, the development of DNA–chitosan nanoparticles (NPs) has gained significant interest for their potential in nucleic acid delivery, especially as non-viral gene delivery systems and for embedding linear DNA fragments, as well as gene delivery to the lung. This study explored an easy polyelectrolyte complexing preparation of linear DNA-loaded chitosan nanoparticles. Among the different formulations of nanoparticles prepared, the optimal one exhibited a size of approximately 290 nm, an encapsulation efficiency of 86% and a zeta potential of 25 mV. Additionally, this study examined how the concentration of DNA in solution influenced nanoparticle formation, encapsulation efficiency and particle size. In particular, transient transfection of the chitosan–linear DNA fragment complex, encoding for green fluorescent protein (GFP), was conducted in human pulmonary distal lung cells (NCI-H441 cells), demonstrating successful cellular internalization and protein expression. These studies highlight the potential of DNA–chitosan NPs in nucleic acid delivery, particularly for pulmonary applications. Future works will focus on formulating the achieved carrier into an inhalable dosage form to improve its translational application. Full article

Lung inflammation is a pivotal event in the pathogenesis of acute lung injury. Heme oxygenase-1 (HO-1) is a key antioxidant enzyme that could be induced by kaempferol (KPR) and exerts anti-inflammatory effects. However, the molecular mechanisms of KPR-mediated HO-1 expression and its effects on inflammatory responses remain unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). This study aimed to verify the relationship between HO-1 expression and KPR treatment in both in vitro and in vivo models. HO-1 expression was determined by real time-PCR, Western blotting, and promoter reporter analyses. The signaling components were investigated by using pharmacological inhibitors or specific siRNAs. Chromatin immunoprecipitation (ChIP) assay was performed to investigate the interaction between nuclear factor erythroid-2-related factor (Nrf2) and antioxidant response elements (ARE) binding site of HO-1 promoter. The effect of KPR on monocytes (THP-1) binding to HPAEpiCs challenged with lipopolysaccharides (LPS) was determined by adhesion assay. We found that KPR-induced HO-1 level attenuated the LPS-induced intercellular cell adhesion protein 1 (ICAM-1) expression in HPAEpiCs. KPR-induced HO-1 mRNA and protein expression also attenuated ICAM-1 expression in mice. Tin protoporphyrin (SnPP)IX reversed the inhibitory effects of KPR in HPAEpiCs. In addition, in HPAEpiCs, KPR-induced HO-1 expression was abolished by both pretreating with the inhibitor of NADPH oxidase (NOX, apocynin (APO)), reactive oxygen species (ROS) (N-acetyl-L-cysteine (NAC)), Src (Src kinase inhibitor II (Srci II)), Pyk2 (PF431396), protein kinase C (PKC)α (Gö6976), p38 mitogen-activated protein kinase (MAPK) inhibitor (p38i) VIII, or c-Jun N-terminal kinases (JNK)1/2 (SP600125) and transfection with their respective siRNAs. The transcription of the homx1 gene was enhanced by Nrf2 activated by JNK1/2 and p38α MAPK. The binding activity between Nrf2 and HO-1 promoter was attenuated by APO, NAC, Srci II, PF431396, or Gö6983. KPR-mediated NOX/ROS/c-Src/Pyk2/PKCα/p38α MAPK and JNK1/2 activate Nrf2 to bind with ARE on the HO-1 promoter and induce HO-1 expression, which further suppresses the LPS-mediated inflammation in HPAEpiCs. Thus, KPR exerts a potential strategy to protect against pulmonary inflammation via upregulation of the HO-1. Full article

In this work, we propose chitosan (CS)-based nanocapsules (NCs) for pulmonary gene delivery. Hyaluronic acid (HA) was incorporated in the NCs composition (HA/CS NCs) aiming to promote gene transfection in the lung epithelium. NCs were loaded with a model plasmid (pCMV-βGal) to easily evaluate their transfection capacity. The plasmid encapsulation efficiencies were of approx. 90%. To facilitate their administration to the lungs, the plasmid-loaded NCs were microencapsulated in mannitol (Ma) microspheres (MS) using a simple spray-drying technique, obtaining dry powders of adequate properties. In vivo, the MS reached the deep lung, where the plasmid-loaded CS-based NCs were released and transfected the alveolar cells more homogeneously than the control formulation of plasmid directly microencapsulated in Ma MS. The HA-containing formulation achieved the highest transfection efficiency, in a more extended area and more homogeneously distributed than the rest of tested formulations. The new micro-nanostructured platform proposed in this work represents an efficient strategy for the delivery of genetic material to the lung, with great potential for the treatment of genetic lung diseases. Full article

The pulmonary endothelium is dysfunctional in chronic obstructive pulmonary disease (COPD), a known risk factor for lung cancer. The pulmonary endothelium is altered in emphysema, which is disproportionately affected by cancers. Gene and microRNA expression differs between COPD and non-COPD lung. We hypothesised that the alteration in microRNA expression in the pulmonary endothelium contributes to its dysfunction. A total of 28 patients undergoing pulmonary resection were recruited and endothelial cells were isolated from healthy lung and tumour. MicroRNA expression was compared between COPD and non-COPD patients. Positive findings were confirmed by quantitative polymerase chain reaction (qPCR). Assays assessing angiogenesis and cellular migration were conducted in Human Umbilical Vein Endothelial Cells (n = 3–4) transfected with microRNA mimics and compared to cells transfected with negative control RNA. Expression of miR-181b-3p, miR-429 and miR-23c (all p < 0.05) was increased in COPD. Over-expression of miR-181b-3p was associated with reduced endothelial sprouting (p < 0.05). miR-429 was overexpressed in lung cancer as well and exhibited a reduction in tubular formation. MicroRNA-driven changes in the pulmonary endothelium thus represent a novel mechanism driving emphysema. These processes warrant further study to determine if they may be therapeutic targets in COPD and lung cancer. Full article

Lung cancer is a worldwide prevalent malignancy. This disease has a low survival rate due to diagnosis at a late stage challenged by the involvement of metastatic sites. Non-small-cell lung cancer (NSCLC) is presented in 85% of cases. The last decade has experienced substantial advancements in scientific research, leading to a novel targeted therapeutic approach. The newly developed pharmaceutical agents are aimed towards specific mutations, detected in individual patients inflicted by lung cancer. These drugs have longer and improved response rates compared to traditional chemotherapy. Recent studies were able to identify rare mutations found in pulmonary tumors. Among the gene alterations detected were mesenchymal epithelial transition factor (MET), human epidermal growth factor 2 (HER2), B-type Raf kinase (BRAF), c-ROS proto-oncogene (ROS1), rearranged during transfection (RET) and neurotrophic tyrosine kinase (NTRK). Ongoing clinical trials are gaining insight onto possible first and second lines of medical treatment options intended to enable progression-free survival to lung cancer patients. Full article

The aim of this study is to explore the role of microRNAs (miR)-21/23a/146a/150/155 targeting the toll-like receptor pathway in active tuberculosis (TB) disease and latent TB infection (LTBI). Gene expression levels of the five miRs and predicted target genes were assessed in peripheral blood mononuclear cells from 46 patients with active pulmonary TB, 15 subjects with LTBI, and 17 non-infected healthy subjects (NIHS). THP-1 cell lines were transfected with miR-23a-3p mimics under stimuli with Mycobacterium TB-specific antigens. Both miR-155-5p and miR-150-5p gene expressions were decreased in the active TB group versus the NIHS group. Both miR-23a-3p and miR-146a-5p gene expressions were decreased in active TB patients with high bacterial burden versus those with low bacterial burden or control group (LTBI + NIHS). TLR2, TLR4, and interleukin (IL)10 gene expressions were all increased in active TB versus NIHS group. MiR-23a-3p mimic transfection reversed ESAT6-induced reduction of reactive oxygen species generation, and augmented ESAT6-induced late apoptosis and phagocytosis, in association with down-regulations of the predicted target genes, including tumor necrosis factor (TNF)-α, TLR4, TLR2, IL6, IL10, Notch1, IL6R, BCL2, TGF-β1, SP1, and IRF1. In conclusion, the down-regulation of miR-23a-3p in active TB patients with high bacterial burden inhibited mononuclear cell function and phagocytosis through TLR4/TNF-α/TGF-β1/IL-10 signaling via targeting IRF1/SP1. Full article

Pathogenic variants have been identified in 85% of heritable pulmonary arterial hypertension (PAH) patients. These variants were mainly located in the bone morphogenetic protein receptor 2 (BMPR2) gene. However, the penetrance of BMPR2 variants was reduced leading to a disease manifestation in only 30% of carriers. In these PAH patients, further modifiers such as additional pathogenic BMPR2 promoter variants could contribute to disease manifestation. Therefore, the aim of this study was to identify BMPR2 promoter variants in PAH patients and to analyze their transcriptional effect on gene expression and disease manifestation. BMPR2 promoter variants were identified in PAH patients and cloned into plasmids. These were transfected into human pulmonary artery smooth muscle cells to determine their respective transcriptional activity. Nine different BMPR2 promoter variants were identified in seven PAH families and three idiopathic PAH patients. Seven of the variants (c.-575A>T, c.-586dupT, c.-910C>T, c.-930_-928dupGGC, c.-933_-928dupGGCGGC, c.-930_-928delGGC and c.-1141C>T) led to a significantly decreased transcriptional activity. This study identified novel BMPR2 promoter variants which may affect BMPR2 gene expression in PAH patients. They could contribute to disease manifestations at least in some families. Further studies are needed to investigate the frequency of BMPR2 promoter variants and their impact on penetrance and disease manifestation. Full article

The present study investigated a pulmonary delivery system of plasmid DNA (pDNA) and its application to melanoma DNA vaccines. pCMV-Luc, pEGFP-C1, and pZsGreen were used as a model pDNA to evaluate transfection efficacy after inhalation in mice. Naked pDNA and a ternary complex, consisting of pDNA, dendrigraft poly-l-lysine (DGL), and γ-polyglutamic acid (γ-PGA), both showed strong gene expression in the lungs after inhalation. The transgene expression was detected in alveolar macrophage-rich sites by observation using multi-color deep imaging. On the basis of these results, we used pUb-M, which expresses melanoma-related antigens (ubiquitinated murine melanoma gp100 and tyrosinase-related protein 2 (TRP2) peptide epitopes), as DNA vaccine for melanoma. The inhalation of naked pUb-M and its ternary complex significantly inhibited the metastasis of B16-F10 cells, a melanoma cell line, in mice. The levels of the inflammatory cytokines, such as TNF-α, IFN-γ, and IL-6, which enhance Th1 responses, were higher with the pUb-M ternary complex than with naked pUb-M and pEGFP-C1 ternary complex as control. In conclusion, we clarified that the inhalation of naked pDNA as well as its ternary complex are a useful technique for cancer vaccination. Full article

Understanding the in vivo fate of lipoplex, which is composed of cationic liposomes and DNA, is an important issue toward gene therapy. In disease conditions, the fate of lipoplex might change compared with the normal condition. Here, we examined the contribution of interaction with serum components to in vivo transfection using lipoplex in hepatitis mice. Prior to administration, lipoplex was incubated with serum or albumin. In the liver, the interaction with albumin enhanced gene expression in hepatitis mice, while in the lung, the interaction with serum or albumin enhanced it. In normal mice, the interaction with albumin did not enhance hepatic and pulmonary gene expression. Furthermore, hepatic and pulmonary gene expression levels of albumin-interacted lipoplex were correlated with serum transaminases in hepatitis mice. The albumin interaction increased the hepatic accumulation of lipoplex and serum tumor necrosis factor-α level. We suggest that the interaction with albumin enhanced the inflammation level after the administration of lipoplex in hepatitis mice. Consequently, the enhancement of the inflammation level might enhance the gene expression level. Information obtained in the current study will be valuable toward future clinical application of the lipoplex. Full article

Antisense Oligonucleotides (ASOs) are an emerging drug class in gene modification. In our study we developed a safe, stable, and effective ASO drug candidate in locked nucleic acid (LNA)-gapmer design, targeting TGFβ receptor II (TGFBR2) mRNA. Discovery was performed as a process using state-of-the-art library development and screening. We intended to identify a drug candidate optimized for clinical development, therefore human specificity and gymnotic delivery were favored by design. A staggered process was implemented spanning in-silico-design, in-vitro transfection, and in-vitro gymnotic delivery of small batch syntheses. Primary in-vitro and in-vivo toxicity studies and modification of pre-lead candidates were also part of this selection process. The resulting lead compound NVP-13 unites human specificity and highest efficacy with lowest toxicity. We particularly focused at attenuation of TGFβ signaling, addressing both safety and efficacy. Hence, developing a treatment to potentially recondition numerous pathological processes mediated by elevated TGFβ signaling, we have chosen to create our data in human lung cell lines and human neuronal stem cell lines, each representative for prospective drug developments in pulmonary fibrosis and neurodegeneration. We show that TGFBR2 mRNA as a single gene target for NVP-13 responds well, and that it bears great potential to be safe and efficient in TGFβ signaling related disorders. Full article

Mevastatin (MVS) has been previously shown to induce heme oxygenase (HO)-1 expression through Nox/ROS-dependent PDGFRα/PI3K/Akt/Nrf2/ARE axis in human pulmonary alveolar epithelial cells (HPAEpiCs). However, alternative signaling pathways might involve in MVS-induced HO-1 expression. We found that tumor necrosis factor α (TNFα) induced vascular cell adhesion protein 1 (VCAM-1) expression and NF-κB p65 phosphorylation which were attenuated by pretreatment with MVS via up-regulation of HO-1, determined by Western blot and real-time qPCR. TNFα-induced VCAM-1 expression was attenuated by an NF-κB inhibitor, Bay117082. The inhibitory effects of MVS were reversed by tin protoporphyrin (SnPP)IX (an inhibitor of HO-1 activity). In addition, pretreatment with the inhibitor of pan-Protein kinase C (PKC) (GF109203X), PKCα (Gö6983), Pyk2 (PF431396), p38α MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA), and transfection with their respective siRNAs abolished MVS-induced HO-1 expression in HPAEpiCs. c-Jun (one of AP-1 subunits) was activated by PKCα, Pyk2, p38α MAPK, and JNK1/2, which turned on the transcription of the homx1 gene. The interaction between c-Jun and HO-1 promoter was confirmed by a chromatin immunoprecipitation (ChIP) assay, which was attenuated by these pharmacological inhibitors. These results suggested that MVS induces AP-1/HO-1 expression via PKCα/Pyk2/p38α MAPK- or JNK1/2-dependent c-Jun activation, which further binds with AP-1-binding site on HO-1 promoter and suppresses the TNFα-mediated inflammatory responses in HPAEpiCs. Thus, upregulation of the AP-1/HO-1 system by MVS exerts a potentially therapeutic strategy to protect against pulmonary inflammation. Full article

Microvascular endothelial cells (ECs) represent the primary target cells during human rickettsioses and respond to infection via the activation of immediate–early signaling cascades and the resultant induction of gene expression. As small noncoding RNAs dispersed throughout the genome, microRNAs (miRNAs) regulate gene expression post-transcriptionally to govern a wide range of biological processes. Based on our recent findings demonstrating the involvement of fibroblast growth factor receptor 1 (FGFR1) in facilitating rickettsial invasion into host cells and published reports suggesting miR-424 and miR-503 as regulators of FGF2/FGFR1, we measured the expression of miR-424 and miR-503 during R. conorii infection of human dermal microvascular endothelial cells (HMECs). Our results revealed a significant decrease in miR-424 and miR-503 expression in apparent correlation with increased expression of FGF2 and FGFR1. Considering the established phenomenon of endothelial heterogeneity and pulmonary and cerebral edema as the prominent pathogenic features of rickettsial infections, and significant pathogen burden in the lungs and brain in established mouse models of disease, we next quantified miR-424 and miR-503 expression in pulmonary and cerebral microvascular ECs. Again, R. conorii infection dramatically downregulated both miRNAs in these tissue-specific ECs as early as 30 min post-infection in correlation with higher FGF2/FGFR1 expression. Changes in the expression of both miRNAs and FGF2/FGFR1 were next confirmed in a mouse model of R. conorii infection. Furthermore, miR-424 overexpression via transfection of a mimic into host ECs reduced the expression of FGF2/FGFR1 and gave a corresponding decrease in R. conorii invasion, while an inhibitor of miR-424 had the expected opposite effect. Together, these findings implicate the rickettsial manipulation of host gene expression via regulatory miRNAs to ensure efficient cellular entry as the critical requirement to establish intracellular infection. Full article

Fibroblasts/myofibroblasts are the key effector cells responsible for excessive extracellular matrix (ECM) deposition and fibrosis progression in both idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) patient lungs, thus it is critical to understand the transcriptomic and proteomic programs underlying their fibrogenic activity. We conducted the first integrative analysis of the fibrotic programming in these cells at the levels of gene and microRNA (miRNA) expression, as well as deposited ECM protein to gain insights into how fibrotic transcriptional programs culminate in aberrant ECM protein production/deposition. We identified messenger RNA (mRNA), miRNA, and deposited matrisome protein signatures for IPF and SSc fibroblasts obtained from lung transplants using next-generation sequencing and mass spectrometry. SSc and IPF fibroblast transcriptional signatures were remarkably similar, with enrichment of WNT, TGF-β, and ECM genes. miRNA-seq identified differentially regulated miRNAs, including downregulation of miR-29b-3p, miR-138-5p and miR-146b-5p in disease fibroblasts and transfection of their mimics decreased expression of distinct sets of fibrotic signature genes as assessed using a Nanostring fibrosis panel. Finally, proteomic analyses uncovered a distinct “fibrotic” matrisome profile deposited by IPF and SSc fibroblasts compared to controls that highlights the dysregulated ECM production underlying their fibrogenic activities. Our comprehensive analyses of mRNA, miRNA, and matrisome proteomic profiles in IPF and SSc lung fibroblasts revealed robust fibrotic signatures at both the gene and protein expression levels and identified novel fibrogenesis-associated miRNAs whose aberrant downregulation in disease fibroblasts likely contributes to their fibrotic and ECM gene expression. Full article

Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Additionally, when established as an episome, they show a very high mitotic stability. In the present study we tested the idea that addition of an S/MAR element to a CFTR (cystic fibrosis transmembrane conductance regulator) expression vector, may allow the establishment of a CFTR episome in bronchial epithelial cells. Starting from the observation that the S/MAR vector pEPI-EGFP (enhanced green fluorescence protein) is maintained as an episome in human bronchial epithelial cells, we assembled the CFTR vector pBQ-S/MAR. This vector, transfected in bronchial epithelial cells with mutated CFTR, supported long term wt CFTR expression and activity, which in turn positively impacted on the assembly of tight junctions in polarized epithelial cells. Additionally, the recovery of intact pBQ-S/MAR, but not the parental vector lacking the S/MAR element, from transfected cells after extensive proliferation, strongly suggested that pBQ-S/MAR was established as an episome. These results add a new element, the S/MAR, that can be considered to improve the persistence and safety of gene therapy vectors for cystic fibrosis pulmonary disease. Full article