PAI-1 Deficiency Drives Pulmonary Vascular Smooth Muscle Remodeling and Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but its role in PAH is unsettled. Here, we report that: (1) PAI-1 is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared to controls; (2) PAI-1-/- mice spontaneously develop pulmonary vascular remodeling associated with up-regulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and (3) pharmacological inhibition of uPA in human PAH PASMCs suppresses pro-proliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that down-regulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent up-regulation of mTOR and TGF-b signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH.

Mice infected with Mycobacterium tuberculosis are resistant to acute disease caused by secondary infection with SARS-CoV-2.

Mycobacterium tuberculosis (Mtb) and SARS-CoV-2 (CoV2) are the leading causes of death due to infectious disease. Although Mtb and CoV2 both cause serious and sometimes fatal respiratory infections, the effect of Mtb infection and its associated immune response on secondary infection with CoV2 is unknown. To address this question we applied two mouse models of COVID19, using mice which were chronically infected with Mtb. In both model systems, Mtb-infected mice were resistant to the pathological consequences of secondary CoV2 infection, and CoV2 infection did not affect Mtb burdens. Single cell RNA sequencing of coinfected and monoinfected lungs demonstrated the resistance of Mtb-infected mice is associated with expansion of T and B cell subsets upon viral challenge. Collectively, these data demonstrate that Mtb infection conditions the lung environment in a manner that is not conducive to CoV2 survival.

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics.

Identifying protein-protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an "abortive" biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.

Saturation Transfer Difference NMR and Molecular Docking Interaction Study of Aralkyl-Thiodigalactosides as Potential Inhibitors of the Human-Galectin-3 Protein.

Human Galectin-3 (hGal-3) is a protein that selectively binds to ß-galactosides and holds diverse roles in both normal and pathological circumstances. Therefore, targeting hGal-3 has become a vibrant area of research in the pharmaceutical chemistry. As a step towards the development of novel hGal-3 inhibitors, we synthesized and investigated derivatives of thiodigalactoside (TDG) modified with different aromatic substituents. Specifically, we describe a high-yielding synthetic route of thiodigalactoside (TDG); an optimized procedure for the synthesis of the novel 3,3'-di-O-(quinoline-2-yl)methyl)-TDG and three other known, symmetric 3,3'-di-O-TDG derivatives ((naphthalene-2yl)methyl, benzyl, (7-methoxy-2H-1-benzopyran-2-on-4-yl)methyl). In the present study, using competition Saturation Transfer Difference (STD) NMR spectroscopy, we determined the dissociation constant (Kd) of the former three TDG derivatives produced to characterize the strength of the interaction with the target protein (hGal-3). Based on the Kd values determined, the (naphthalen-2-yl)methyl, the (quinolin-2-yl)methyl and the benzyl derivatives bind to hGal-3 94, 30 and 24 times more strongly than TDG. Then, we studied the binding modes of the derivatives in silico by molecular docking calculations. Docking poses similar to the canonical binding modes of well-known hGal-3 inhibitors have been found. However, additional binding forces, cation-π interactions between the arginine residues in the binding pocket of the protein and the aromatic groups of the ligands, have been established as significant features. Our results offer a molecular-level understanding of the varying affinities observed among the synthesized thiodigalactoside derivatives, which can be a key aspect in the future development of more effective ligands of hGal-3.

Towards Machine Learning in Heterogeneous Catalysis-A Case Study of 2,4-Dinitrotoluene Hydrogenation.

Utilization of multivariate data analysis in catalysis research has extraordinary importance. The aim of the MIRA21 (MIskolc RAnking 21) model is to characterize heterogeneous catalysts with bias-free quantifiable data from 15 different variables to standardize catalyst characterization and provide an easy tool to compare, rank, and classify catalysts. The present work introduces and mathematically validates the MIRA21 model by identifying fundamentals affecting catalyst comparison and provides support for catalyst design. Literature data of 2,4-dinitrotoluene hydrogenation catalysts for toluene diamine synthesis were analyzed by using the descriptor system of MIRA21. In this study, exploratory data analysis (EDA) has been used to understand the relationships between individual variables such as catalyst performance, reaction conditions, catalyst compositions, and sustainable parameters. The results will be applicable in catalyst design, and using machine learning tools will also be possible.

Development of Palladium and Platinum Decorated Granulated Carbon Nanocomposites for Catalytic Chlorate Elimination.

Granulated carbon nanotube-supported palladium and platinum-containing catalysts were developed. By using these, remarkable catalytic activity was achieved in chlorate ion hydrogenation. Nitrogen-doped bamboo-like carbon nanotubes (N-BCNTs) loaded gel beads were prepared by using Ca2+, Ni2+ or Fe3+ ions as precursors for cross-linking of sodium alginate. The gel beads were carbonized at 800 °C, and these granulated carbon nanocomposites (GCNC) were used as supports to prepare palladium and platinum-containing catalysts. All in all, three catalysts were developed and, in each case, >99 n/n% chlorate conversion was reached in the aqueous phase by using the Pd-Pt containing GCNCs, moreover, these systems retained their catalytic activity even after repeated use.

A Fucosylated Lactose-Presenting Tetravalent Glycocluster Acting as a Mutual Ligand of Pseudomonas aeruginosa Lectins A (PA-IL) and B (PA-IIL)-Synthesis and Interaction Studies.

The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic human pathogen associated with cystic fibrosis. P. aeruginosa produces two soluble lectins, the d-galactose-specific lectin PA-IL (LecA) and the l-fucose-specific lectin PA-IIL (LecB), among other virulence factors. These lectins play an important role in the adhesion to host cells and biofilm formation. Moreover, PA-IL is cytotoxic to respiratory cells in the primary culture. Therefore, these lectins are promising therapeutic targets. Specifically, carbohydrate-based compounds could inhibit their activity. In the present work, a 3-O-fucosyl lactose-containing tetravalent glycocluster was synthesized and utilized as a mutual ligand of galactophilic and fucophilic lectins. Pentaerythritol equipped with azido ethylene glycol-linkers was chosen as a multivalent scaffold and the glycocluster was constructed by coupling the scaffold with propargyl 3-O-fucosyl lactoside using an azide-alkyne 1,3-dipolar cycloaddition reaction. The interactions between the glycocluster and PA-IL or PA-IIL were investigated by isothermal titration microcalorimetry and saturation transfer difference NMR spectroscopy. These results may assist in the development of efficient anti-adhesion therapy for the treatment of a P. aeruginosa infection.

Epigenetic Regulation of Endothelial Dysfunction and Inflammation in Pulmonary Arterial Hypertension.

Once perceived as a disorder treated by vasodilation, pulmonary artery hypertension (PAH) has emerged as a pulmonary vascular disease with severe endothelial cell dysfunction. In the absence of a cure, many studies seek to understand the detailed mechanisms of EC regulation to potentially create more therapeutic options for PAH. Endothelial dysfunction is characterized by complex phenotypic changes including unchecked proliferation, apoptosis-resistance, enhanced inflammatory signaling and metabolic reprogramming. Recent studies have highlighted the role of epigenetic modifications leading to pro-inflammatory response pathways, endothelial dysfunction, and the progression of PAH. This review summarizes the existing literature on epigenetic mechanisms such as DNA methylation, histone modifications, and non-coding RNAs, which can lead to aberrant endothelial function. Our goal is to develop a conceptual framework for immune dysregulation and epigenetic changes in endothelial cells in the context of PAH. These studies as well as others may lead to advances in therapeutics to treat this devastating disease.

Toll-like Receptor 3 Is a Therapeutic Target for Pulmonary Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by vascular cell proliferation and endothelial cell apoptosis. TLR3 (Toll-like receptor 3) is a receptor for double-stranded RNA and has been recently implicated in vascular protection. OBJECTIVES: To study the expression and role of TLR3 in PAH and to determine whether a TLR3 agonist reduces pulmonary hypertension in preclinical models. METHODS: Lung tissue and endothelial cells from patients with PAH were investigated by polymerase chain reaction, immunofluorescence, and apoptosis assays. TLR3-/- and TLR3+/+ mice were exposed to chronic hypoxia and SU5416. Chronic hypoxia or chronic hypoxia/SU5416 rats were treated with the TLR3 agonist polyinosinic/polycytidylic acid (Poly[I:C]). MEASUREMENTS AND MAIN RESULTS: TLR3 expression was reduced in PAH patient lung tissue and endothelial cells, and TLR3-/- mice exhibited more severe pulmonary hypertension following exposure to chronic hypoxia/SU5416. TLR3 knockdown promoted double-stranded RNA signaling via other intracellular RNA receptors in endothelial cells. This was associated with greater susceptibility to apoptosis, a known driver of pulmonary vascular remodeling. Poly(I:C) increased TLR3 expression via IL-10 in rat endothelial cells. In vivo, high-dose Poly(I:C) reduced pulmonary hypertension in both rat models in proof-of-principle experiments. In addition, Poly(I:C) also reduced right ventricular failure in established pulmonary hypertension. CONCLUSIONS: Our work identifies a novel role for TLR3 in PAH based on the findings that reduced expression of TLR3 contributes to endothelial apoptosis and pulmonary vascular remodeling.

RNA Signaling in Pulmonary Arterial Hypertension-A Double-Stranded Sword.

Recognition of and response to pathogens and tissue injury is driven by the innate immune system via activation of pattern recognition receptors. One of the many patterns recognized is RNA and, while several receptors bind RNA, Toll-like receptor 3 (TLR3) is well placed for initial recognition of RNA molecules due to its localization within the endosome. There is a growing body of work describing a role for TLR3 in maintenance of vascular homeostasis. For example, TLR3 deficiency has been shown to play repair and remodeling roles in the systemic vasculature and in lung parenchyma. A hallmark of pulmonary arterial hypertension (PAH) is pulmonary vascular remodeling, yet drivers and triggers of this remodeling remain incompletely understood. Based on its role in the systemic vasculature, our group discovered reduced endothelial TLR3 expression in PAH and revealed a protective role for a TLR3 agonist in rodent models of pulmonary hypertension. This review will provide an overview of RNA signaling in the vasculature and how it relates to PAH pathobiology, including whether targeting double-stranded RNA signaling is a potential treatment option for PAH.

Impact of parenchymal loss on renal function after laparoscopic partial nephrectomy under warm ischemia.

PURPOSE: To elucidate the impact of renal parenchymal loss and the ischemic reperfusion injury (RI) on the renal function after laparoscopic partial nephrectomy (LPN) under warm ischemia (WI). METHODS: Thirty-five patients with a single polar renal mass ≤4 cm and normal contralateral kidney underwent LPN. Transperitoneal LPN with WI using en bloc hilar occlusion was performed. The total differential renal function (T-DRF) using 99mTc-dimercaptosuccinic acid was evaluated preoperatively and postoperatively over a period of 1 year. A special region of interest (ROI) was selected on the non-tumorous pole of the involved kidney, and was compared with the same ROI in the contralateral kidney. The latter comparison was defined as partial differential renal function (P-DRF). Any postoperative decline in the P-DRF of the operated kidney was attributed to the RI. Subtraction of the P-DRF decline from the T-DRF decline was attributed to the parenchymal loss caused by the resection of the tumor and suturing of the normal parenchyma. RESULTS: The mean WI time was 22 min, and the mean weight of resected specimen was 18 g. The mean postoperative eGFR declined to 87 ml/min/1.73 m2 from its baseline mean value of 97 ml/min/1.73 m2 (p value = 0.075). Mean postoperative T-DRF and P-DRF of the operated kidney declined by 7 and 3 %, respectively. CONCLUSIONS: After LPN of small renal mass, decline in renal function is primarily attributed to parenchymal loss caused by tumor resection and suturing of the normal parenchyma rather than the RI.

IL-32 promotes angiogenesis.

IL-32 is a multifaceted cytokine with a role in infections, autoimmune diseases, and cancer, and it exerts diverse functions, including aggravation of inflammation and inhibition of virus propagation. We previously identified IL-32 as a critical regulator of endothelial cell (EC) functions, and we now reveal that IL-32 also possesses angiogenic properties. The hyperproliferative ECs of human pulmonary arterial hypertension and glioblastoma multiforme exhibited a markedly increased abundance of IL-32, and, significantly, the cytokine colocalized with integrin αVß3. Vascular endothelial growth factor (VEGF) receptor blockade, which resulted in EC hyperproliferation, increased IL-32 three-fold. Small interfering RNA-mediated silencing of IL-32 negated the 58% proliferation of ECs that occurred within 24 h in scrambled-transfected controls. Reduction of IL-32 neither affected apoptosis (insignificant changes in Bak-1, Bcl-2, Bcl-xL, lactate dehydrogenase, annexin V, and propidium iodide) nor VEGF or TGF-ß levels, but siIL-32-transfected adult and neonatal ECs produced up to 61% less NO, IL-8, and matrix metalloproteinase-9, and up to 3-fold more activin A and endostatin. In coculture-based angiogenesis assays, IL-32γ dose-dependently increased tube formation up to 3-fold; an αVß3 inhibitor prevented this activity and reduced IL-32γ-induced IL-8 by 85%. In matrigel plugs loaded with IL-32γ, VEGF, or vehicle and injected into live mice, we observed the anticipated VEGF-induced increase in neocapillarization (8-fold versus vehicle), but unexpectedly, IL-32γ was equally angiogenic. A second signal such as IFN-γ was required to render cells responsive to exogenous IL-32γ; importantly, this was confirmed using a completely synthetic preparation of IL-32γ. In summary, we add angiogenic properties that are mediated by integrin αVß3 but VEGF-independent to the portfolio of IL-32, implicating a role for this versatile cytokine in pulmonary arterial hypertension and neoplastic diseases.

Iloprost reverses established fibrosis in experimental right ventricular failure.

Prostacyclin and its analogues improve cardiac output and functional capacity in patients with pulmonary arterial hypertension (PAH); however, the underlying mechanism is not fully understood. We hypothesised that prostanoids have load-independent beneficial effects on the right ventricle (RV). Angio-obliterative PAH and RV failure were induced in rats with a single injection of SU5416 followed by 4 weeks of exposure to hypoxia. Upon confirmation of RV dysfunction and PAH, rats were randomised to 0.1 µg·kg(-1) nebulised iloprost or drug-free vehicle, three times daily for 2 weeks. RV function and treadmill running time were evaluated pre- and post-iloprost/vehicle treatment. Pulmonary artery banded rats were treated 8 weeks after surgery to allow for significant RV hypertrophy. Inhaled iloprost significantly improved tricuspid annulus plane systolic excursion and increased exercise capacity, while mean pulmonary artery pressure and the percentage of occluded pulmonary vessels remained unchanged. Rats treated with iloprost had a striking reduction in RV collagen deposition, procollagen mRNA levels and connective tissue growth factor expression in both SU5416/hypoxia and pulmonary artery banded rats. In vitro, cardiac fibroblasts treated with iloprost showed a reduction in transforming growth factor (TGF)-ß1-induced connective tissue growth factor expression, in a protein kinase A-dependent manner. Iloprost decreased TGF-ß1-induced procollagen mRNA expression as well as cardiac fibroblast activation and migration. Iloprost significantly induced metalloproteinase-9 gene expression and activity and increased the expression of autophagy genes associated with collagen degradation. Inhaled iloprost improves RV function and reverses established RV fibrosis partially by preventing collagen synthesis and by increasing collagen turnover.

Glycosyltransferases and glycosaminoglycans in bleomycin and transforming growth factor-ß1-induced pulmonary fibrosis.

Glycosaminoglycan (GAG) chains of proteoglycans (PGs) play important roles in fibrosis through cell-matrix interactions and growth factor binding in the extracellular matrix. We investigated the expression and regulation of PG core protein (versican) and key enzymes (xylosyltransferase [XT]-I, ß1,3-glucuronosyltransferase [GlcAT]-I, chondroitin-4-sulfotransferase [C4ST]) implicated in synthesis and sulfation of GAGs in bleomycin (BLM) and adenovirus-transforming growth factor (TGF)-ß1-induced lung fibrosis in rats. We also studied the role of GlcAT-I or TGF-ß1 and the signaling pathways regulating PG-GAG production in primary lung fibroblasts isolated from saline- or BLM-instilled rats. The mRNA for XT-I, GlcAT-I, C4ST, and versican was increased in the lung 14 days after BLM injury. In vitro studies indicate that fibrotic lung fibroblasts (FLFs) expressed more XT-I, C4ST, and chondroitin sulfate (CS)-GAGs than did normal lung fibroblasts at baseline. TGF-ß1 enhanced the expression of XT-I, C4ST-I, and versican in normal lung fibroblasts, whereas SB203580 or SB431542, by targeting p38 mitogen-activated protein kinase or TGF-ß type-1 receptor/activin receptor-like kinase 5, respectively, attenuated the response to both TGF-ß1 and FLFs on PG-GAG expression. Neutralizing anti-TGF-ß1 antibody abrogated FLF-conditioned medium-stimulated expression of XT-I, GlcAT-I, versican, and CS-GAG. Forced expression of TGF-ß1 in vivo enhanced versican, XT-I, GlcAT-I, and C4ST-I expression and PG-GAG deposition in rat lungs. Finally, induced expression of GlcAT-I gene in rat lung fibroblasts increased GAG synthesis by these cells. Together, our results provide new insights into the basis for increased PG-GAG deposition in lung fibrosis; inhibition of TGF-ß1-mediated or fibrosis-induced PG-GAG production by activin receptor-like kinase 5/p38 inhibitors may contribute to antifibrotic activity.