Bmi-1 alleviates adventitial fibroblast senescence by eliminating ROS in pulmonary hypertension

Abstract Objectives Pulmonary hypertension (PH) is a life-threatening progressive disease with high mortality in the elderly. However, the pathogenesis of PH has not been fully understood and there is no effective therapy to reverse the disease process. This study aims to determine whether cellular senescence is involved in the development of PH. Methods The rat PH model was established by intraperitoneal injection of monocrotaline and evaluated by pulmonary arteriole wall thickness and right ventricular hypertrophy index. Human lung fibroblasts (HLFs) were treated with CoCl2 or hypoxia to induce cellular senescence in vitro. SA-β-gal staining and the changes of senescent markers were used to examine cellular senescence. The molecular mechanism of cellular senescence was further explored by detecting reactive oxygen species (ROS) levels and culturing cells with a conditioned medium. Results We revealed the cellular senescence of pulmonary adventitial fibroblasts in vivo in the rat PH model. The expression of Bmi-1, an important regulator of senescence, was decreased in the lungs of PH rats and localized in adventitial fibroblasts. The in vitro experiments showed that p16 expression was increased while Bmi-1 expression was decreased after CoCl2 treatment in HLFs. Mechanistically, Bmi-1 could alleviate CoCl2-induced HLFs senescence by eliminating ROS which further promoted the proliferation of pulmonary artery smooth muscle cells by paracrine mode of action of HLFs. Conclusion Bmi-1 alleviates the cellular senescence of pulmonary fibroblasts in PH, which expands the pathogenesis of PH and provides a theoretical basis for targeting senescent cells in the treatment of PH.

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Microenvironmental Regulation of Macrophage Transcriptomic and Metabolomic Profiles in Pulmonary Hypertension

Frontiers in Immunology ◽

10.3389/fimmu.2021.640718 ◽

2021 ◽

Vol 12 ◽

Author(s):

Min Li ◽

Suzette Riddle ◽

Sushil Kumar ◽

Joanna Poczobutt ◽

B. Alexandre McKeon ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Macrophage Polarization ◽

Pulmonary Arteries ◽

Cell Types ◽

Metabolic Reprogramming ◽

Macrophage Phenotype ◽

Inflammatory Monocytes ◽

Adventitial Fibroblasts

The recruitment and subsequent polarization of inflammatory monocytes/macrophages in the perivascular regions of pulmonary arteries is a key feature of pulmonary hypertension (PH). However, the mechanisms driving macrophage polarization within the adventitial microenvironment during PH progression remain unclear. We previously established that reciprocal interactions between fibroblasts and macrophages are essential in driving the activated phenotype of both cell types although the signals involved in these interactions remain undefined. We sought to test the hypothesis that adventitial fibroblasts produce a complex array of metabolites and proteins that coordinately direct metabolomic and transcriptomic re-programming of naïve macrophages to recapitulate the pathophysiologic phenotype observed in PH. Media conditioned by pulmonary artery adventitial fibroblasts isolated from pulmonary hypertensive (PH-CM) or age-matched control (CO-CM) calves were used to activate bone marrow derived macrophages. RNA-Seq and mass spectrometry-based metabolomics analyses were performed. Fibroblast conditioned medium from patients with idiopathic pulmonary arterial hypertension or controls were used to validate transcriptional findings. The microenvironment was targeted in vitro using a fibroblast-macrophage co-culture system and in vivo in a mouse model of hypoxia-induced PH. Both CO-CM and PH-CM actively, yet distinctly regulated macrophage transcriptomic and metabolomic profiles. Network integration revealed coordinated rewiring of pro-inflammatory and pro-remodeling gene regulation in concert with altered mitochondrial and intermediary metabolism in response to PH-CM. Pro-inflammation and metabolism are key regulators of macrophage phenotype in vitro, and are closely related to in vivo flow sorted lung interstitial/perivascular macrophages from hypoxic mice. Metabolic changes are accompanied by increased free NADH levels and increased expression of a metabolic sensor and transcriptional co-repressor, C-terminal binding protein 1 (CtBP1), a mechanism shared with adventitial PH-fibroblasts. Targeting the microenvironment created by both cell types with the CtBP1 inhibitor MTOB, inhibited macrophage pro-inflammatory and metabolic re-programming both in vitro and in vivo. In conclusion, coordinated transcriptional and metabolic reprogramming is a critical mechanism regulating macrophage polarization in response to the complex adventitial microenvironment in PH. Targeting the adventitial microenvironment can return activated macrophages toward quiescence and attenuate pathological remodeling that drives PH progression.

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In vivo assessment of a single adenine mutation in 5′UTR of Endothelin-1 gene in paediatric cases with severe pulmonary hypertension: an observational study

BMC Research Notes ◽

10.1186/s13104-021-05609-5 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Abhishek Kumar ◽

Minati Choudhury ◽

Sakshi Dhingra Batra ◽

Kriti Sikri ◽

Anushree Gupta

Keyword(s):

Pulmonary Hypertension ◽

Congenital Heart ◽

Small Sample Size ◽

Small Sample ◽

Severe Pulmonary Hypertension ◽

Endothelin 1 ◽

Circulating Levels ◽

In Vivo Assessment

Abstract Objective Endothelin-1 plays an important role in the pathogenesis of severe pulmonary hypertension. The + 139 ‘A’, adenine insertion variant in 5′UTR of edn1 gene has been reported to be associated with increased expression of Endothelin-1 in vitro. The aim of present study was to explore the association of this variant with the circulating levels of Endothelin-1 in vivo using archived DNA and plasma samples from 38 paediatric congenital heart disease (cyanotic and acyanotic) patients with severe pulmonary hypertension. Results The plasma Endothelin-1 levels were highly varied ranging from 1.63 to75.16 pg/ml. The + 139 ‘A’ insertion variant in 5′UTR of edn1 was seen in 8 out of 38 cases with only one acyanotic sample demonstrating homozygosity of inserted ‘A’ allele at + 139 site (4A/4A genotype). The plasma Endothelin-1 levels in children with homozygous variant 3A/3A genotype were comparable in cyanotic and acyanotic groups. Lone 4A/4A acyanotic sample had ET-1 levels similar to the median value of ET-1 associated with 3A/3A genotype and was absent in cyanotic group presumably due to deleterious higher ET-1 levels. The discussed observations, limited by the small sample size, are suggestive of homozygous adenine insertion variant posing a risk in cyanotic babies with Severe Pulmonary Hypertension.

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An Evidence-Based Systematic Review of Human Knee Post-Traumatic Osteoarthritis (PTOA): Timeline of Clinical Presentation and Disease Markers, Comparison of Knee Joint PTOA Models and Early Disease Implications

International Journal of Molecular Sciences ◽

10.3390/ijms22041996 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1996 ◽

Cited By ~ 1

Author(s):

Christine M. Khella ◽

Rojiar Asgarian ◽

Judith M. Horvath ◽

Bernd Rolauffs ◽

Melanie L. Hart

Keyword(s):

Synovial Fluid ◽

Knee Joint ◽

Ex Vivo ◽

Disease Process ◽

Early Disease ◽

Knee Trauma ◽

Post Traumatic ◽

Acute Knee

Understanding the causality of the post-traumatic osteoarthritis (PTOA) disease process of the knee joint is important for diagnosing early disease and developing new and effective preventions or treatments. The aim of this review was to provide detailed clinical data on inflammatory and other biomarkers obtained from patients after acute knee trauma in order to (i) present a timeline of events that occur in the acute, subacute, and chronic post-traumatic phases and in PTOA, and (ii) to identify key factors present in the synovial fluid, serum/plasma and urine, leading to PTOA of the knee in 23–50% of individuals who had acute knee trauma. In this context, we additionally discuss methods of simulating knee trauma and inflammation in in vivo, ex vivo articular cartilage explant and in vitro chondrocyte models, and answer whether these models are representative of the clinical inflammatory stages following knee trauma. Moreover, we compare the pro-inflammatory cytokine concentrations used in such models and demonstrate that, compared to concentrations in the synovial fluid after knee trauma, they are exceedingly high. We then used the Bradford Hill Framework to present evidence that TNF-α and IL-6 cytokines are causal factors, while IL-1β and IL-17 are credible factors in inducing knee PTOA disease progresssion. Lastly, we discuss beneficial infrastructure for future studies to dissect the role of local vs. systemic inflammation in PTOA progression with an emphasis on early disease.

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Diabetic Cataract—Pathogenesis, Epidemiology and Treatment

Journal of Ophthalmology ◽

10.1155/2010/608751 ◽

2010 ◽

Vol 2010 ◽

pp. 1-8 ◽

Cited By ~ 154

Author(s):

Andreas Pollreisz ◽

Ursula Schmidt-Erfurth

Keyword(s):

Experimental Studies ◽

Disease Process ◽

Basic Research ◽

Polyol Pathway ◽

Population Based ◽

Diabetic Patients ◽

Diabetic Cataract ◽

Cataract Development

Cataract in diabetic patients is a major cause of blindness in developed and developing countries. The pathogenesis of diabetic cataract development is still not fully understood. Recent basic research studies have emphasized the role of the polyol pathway in the initiation of the disease process. Population-based studies have greatly increased our knowledge concerning the association between diabetes and cataract formation and have defined risk factors for the development of cataract. Diabetic patients also have a higher risk of complications after phacoemulsification cataract surgery compared to nondiabetics. Aldose-reductase inhibitors and antioxidants have been proven beneficial in the prevention or treatment of this sightthreatening condition in in vitro and in vivo experimental studies. This paper provides an overview of the pathogenesis of diabetic cataract, clinical studies investigating the association between diabetes and cataract development, and current treatment of cataract in diabetics.

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Accelerated Repurposing and Drug Development of Pulmonary Hypertension Therapies for COVID-19 Treatment Using an AI-Integrated Biosimulation Platform

Molecules ◽

10.3390/molecules26071912 ◽

2021 ◽

Vol 26 (7) ◽

pp. 1912

Author(s):

Kaushik Chakravarty ◽

Victor G. Antontsev ◽

Maksim Khotimchenko ◽

Nilesh Gupta ◽

Aditya Jagarapu ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Renin Angiotensin System ◽

Mechanistic Modeling ◽

Channel Blockers ◽

Angiotensin System ◽

In Silico Modeling ◽

Site Of Action ◽

Line Of Therapy

The COVID-19 pandemic has reached over 100 million worldwide. Due to the multi-targeted nature of the virus, it is clear that drugs providing anti-COVID-19 effects need to be developed at an accelerated rate, and a combinatorial approach may stand to be more successful than a single drug therapy. Among several targets and pathways that are under investigation, the renin-angiotensin system (RAS) and specifically angiotensin-converting enzyme (ACE), and Ca2+-mediated SARS-CoV-2 cellular entry and replication are noteworthy. A combination of ACE inhibitors and calcium channel blockers (CCBs), a critical line of therapy for pulmonary hypertension, has shown therapeutic relevance in COVID-19 when investigated independently. To that end, we conducted in silico modeling using BIOiSIM, an AI-integrated mechanistic modeling platform by utilizing known preclinical in vitro and in vivo datasets to accurately simulate systemic therapy disposition and site-of-action penetration of the CCBs and ACEi compounds to tissues implicated in COVID-19 pathogenesis.

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Calcium channel ITPR2 and mitochondria–ER contacts promote cellular senescence and aging

Nature Communications ◽

10.1038/s41467-021-20993-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Dorian V. Ziegler ◽

David Vindrieux ◽

Delphine Goehrig ◽

Sara Jaber ◽

Guillaume Collin ◽

...

Keyword(s):

Cellular Senescence ◽

Calcium Release ◽

Liver Steatosis ◽

Metabolic Stress ◽

Calcium Release Channel ◽

Release Channel ◽

Age Related ◽

Trisphosphate Receptor

AbstractCellular senescence is induced by stresses and results in a stable proliferation arrest accompanied by a pro-inflammatory secretome. Senescent cells accumulate during aging, promoting various age-related pathologies and limiting lifespan. The endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) calcium-release channel and calcium fluxes from the ER to the mitochondria are drivers of senescence in human cells. Here we show that Itpr2 knockout (KO) mice display improved aging such as increased lifespan, a better response to metabolic stress, less immunosenescence, as well as less liver steatosis and fibrosis. Cellular senescence, which is known to promote these alterations, is decreased in Itpr2 KO mice and Itpr2 KO embryo-derived cells. Interestingly, ablation of ITPR2 in vivo and in vitro decreases the number of contacts between the mitochondria and the ER and their forced contacts induce premature senescence. These findings shed light on the role of contacts and facilitated exchanges between the ER and the mitochondria through ITPR2 in regulating senescence and aging.

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A Tissue-Engineered Tracheobronchial In Vitro Co-Culture Model for Determining Epithelial Toxicological and Inflammatory Responses

Biomedicines ◽

10.3390/biomedicines9060631 ◽

2021 ◽

Vol 9 (6) ◽

pp. 631

Author(s):

Luis Soriano ◽

Tehreem Khalid ◽

Fergal J. O'Brien ◽

Cian O'Leary ◽

Sally-Ann Cryan

Keyword(s):

Epithelial Cells ◽

Inflammatory Responses ◽

Bronchial Epithelial Cells ◽

Lung Fibroblasts ◽

Drug Induced ◽

Bronchial Epithelial ◽

Culture Model ◽

Epithelial Cultures

Translation of novel inhalable therapies for respiratory diseases is hampered due to the lack of in vitro cell models that reflect the complexity of native tissue, resulting in many novel drugs and formulations failing to progress beyond preclinical assessments. The development of physiologically-representative tracheobronchial tissue analogues has the potential to improve the translation of new treatments by more accurately reflecting in vivo respiratory pharmacological and toxicological responses. Herein, advanced tissue-engineered collagen hyaluronic acid bilayered scaffolds (CHyA-B) previously developed within our group were used to evaluate bacterial and drug-induced toxicity and inflammation for the first time. Calu-3 bronchial epithelial cells and Wi38 lung fibroblasts were grown on either CHyA-B scaffolds (3D) or Transwell® inserts (2D) under air liquid interface (ALI) conditions. Toxicological and inflammatory responses from epithelial monocultures and co-cultures grown in 2D or 3D were compared, using lipopolysaccharide (LPS) and bleomycin challenges to induce bacterial and drug responses in vitro. The 3D in vitro model exhibited significant epithelial barrier formation that was maintained upon introduction of co-culture conditions. Barrier integrity showed differential recovery in CHyA-B and Transwell® epithelial cultures. Basolateral secretion of pro-inflammatory cytokines to bacterial challenge was found to be higher from cells grown in 3D compared to 2D. In addition, higher cytotoxicity and increased basolateral levels of cytokines were detected when epithelial cultures grown in 3D were challenged with bleomycin. CHyA-B scaffolds support the growth and differentiation of bronchial epithelial cells in a 3D co-culture model with different transepithelial resistance in comparison to the same co-cultures grown on Transwell® inserts. Epithelial cultures in an extracellular matrix like environment show distinct responses in cytokine release and metabolic activity compared to 2D polarised models, which better mimic in vivo response to toxic and inflammatory stimuli offering an innovative in vitro platform for respiratory drug development.

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Peptide Blocking Self-Polymerization of Extracellular Calcium-Sensing Receptor Attenuates Hypoxia-Induced Pulmonary Hypertension

Hypertension ◽

10.1161/hypertensionaha.120.16712 ◽

2021 ◽

Author(s):

Rui Xiao ◽

Shengquan Luo ◽

Ting Zhang ◽

Yankai Lv ◽

Tao Wang ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Disulfide Bonds ◽

Acute Hypoxia ◽

Extracellular Domain ◽

Left Ventricular ◽

Extracellular Calcium ◽

Calcium Sensing Receptor ◽

Calcium Sensing

Activation of the CaSR (extracellular calcium-sensing receptor) has been recognized as a critical mediator of hypoxia-induced pulmonary hypertension. Preventive targeting of the early initiating phase as well as downstream events after CaSR activation remains unexplored. As a representative of the G protein-coupled receptor family, CaSR polymerizes on cell surface upon stimulation. Immunoblotting together with MAL-PEG technique identified a reactive oxygen species-sensitive CaSR polymerization through its extracellular domain in pulmonary artery smooth muscle cells upon exposure to acute hypoxia. Fluorescence resonance energy transfer screening employing blocking peptides determined that cycteine129/131 residues in the extracellular domain of CaSR formed intermolecular disulfide bonds to promote CaSR polymerization. The monitoring of intracellular Ca 2+ signal highlighted the pivotal role of CaSR polymerization in its activation. In contrast, the blockade of disulfide bonds formation using a peptide decreased both CaSR and hypoxia-induced mitogenic factor expression as well as other hypoxic-related genes in vitro and in vivo and attenuated pulmonary hypertension development in rats. The blocking peptide did not affect systemic arterial oxygenation in vivo but inhibited acute hypoxia-induced pulmonary vasoconstriction. Pharmacokinetic analyses revealed a more efficient lung delivery of peptide by inhaled nebulizer compared to intravenous injection. In addition, the blocking peptide did not affect systemic arterial pressure, body weight, left ventricular function, liver, or kidney function or plasma Ca 2+ level. In conclusion, a peptide blocking CaSR polymerization reduces its hypoxia-induced activation and downstream events leading to pulmonary hypertension and represents an attractive inhaled preventive alternative worthy of further development.

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Comparison of the hemodynamic effects of nitric oxide and endothelium-dependent vasodilators in intact lungs

Journal of Applied Physiology ◽

10.1152/jappl.1990.68.2.735 ◽

1990 ◽

Vol 68 (2) ◽

pp. 735-747 ◽

Cited By ~ 51

Author(s):

S. L. Archer ◽

K. Rist ◽

D. P. Nelson ◽

E. G. DeMaster ◽

N. Cowan ◽

...

Keyword(s):

Nitric Oxide ◽

Methylene Blue ◽

Pulmonary Hypertension ◽

Feedback Inhibition ◽

Pressor Response ◽

Pulmonary Vasculature ◽

Hemodynamic Effects ◽

Isolated Lung

The effects of endothelium-dependent vasodilation on pulmonary vascular hemodynamics were evaluated in a variety of in vivo and in vitro models to determine 1) the comparability of the hemodynamic effects of acetylcholine (ACh), bradykinin (BK), nitric oxide (NO), and 8-bromo-guanosine 3′,5′-cyclic monophosphate (cGMP), 2) whether methylene blue is a useful inhibitor of endothelium-dependent relaxing factor (EDRF) activity in vivo, and 3) the effect of monocrotaline-induced pulmonary hypertension on the responsiveness of the pulmonary vasculature to ACh. In isolated rat lungs, which were preconstricted with hypoxia, ACh, BK, NO, and 8-bromo-cGMP caused pulmonary vasodilation, which was not inhibited by maximum tolerable doses of methylene blue. Methylene blue did not inhibit EDRF activity in any model, despite causing increased pulmonary vascular tone and responsiveness to various constrictor agents. There were significant differences in the hemodynamic characteristics of ACh, BK, and NO. In the isolated lung, BK and NO caused transient decreases of hypoxic vasoconstriction, whereas ACh caused more prolonged vasodilation. Pretreatment of these lungs with NO did not significantly inhibit ACh-induced vasodilation but caused BK to produce vasoconstriction. Tachyphylaxis, which was agonist specific, developed with repeated administration of ACh or BK but not NO. Tachyphylaxis probably resulted from inhibition of the endothelium-dependent vasodilation pathway proximal to NO synthesis, because it could be overcome by exogenous NO. Pretreatment with 8-bromo-cGMP decreased hypoxic pulmonary vasoconstriction and, even when the hypoxic pressor response had largely recovered, subsequent doses of ACh and NO failed to cause vasodilation, although BK produced vasoconstriction. These findings are compatible with the existence of feedback inhibition of the endothelium-dependent relaxation by elevation of cGMP levels. Responsiveness to ACh was retained in lungs with severe monocrotaline-induced pulmonary hypertension. Many of these findings would not have been predicted based on in vitro studies and illustrate the importance for expanding studies of EDRF to in vivo and ex vivo models.

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Glycation of Host Proteins Increases Pathogenic Potential of Porphyromonas gingivalis

International Journal of Molecular Sciences ◽

10.3390/ijms222112084 ◽

2021 ◽

Vol 22 (21) ◽

pp. 12084

Author(s):

Michał Śmiga ◽

John W. Smalley ◽

Paulina Ślęzak ◽

Jason L. Brown ◽

Klaudia Siemińska ◽

...

Keyword(s):

Porphyromonas Gingivalis ◽

Biofilm Formation ◽

Disease Process ◽

Human Keratinocytes ◽

Bacterial Biofilm ◽

Pathogenic Potential ◽

Glycated Proteins ◽

Sds Page

The non-enzymatic addition of glucose (glycation) to circulatory and tissue proteins is a ubiquitous pathophysiological consequence of hyperglycemia in diabetes. Given the high incidence of periodontitis and diabetes and the emerging link between these conditions, it is of crucial importance to define the basic virulence mechanisms employed by periodontopathogens such as Porphyromonas gingivalis in mediating the disease process. The aim of this study was to determine whether glycated proteins are more easily utilized by P. gingivalis to stimulate growth and promote the pathogenic potential of this bacterium. We analyzed the properties of three commonly encountered proteins in the periodontal environment that are known to become glycated and that may serve as either protein substrates or easily accessible heme sources. In vitro glycated proteins were characterized using colorimetric assays, mass spectrometry, far- and near-UV circular dichroism and UV–visible spectroscopic analyses and SDS-PAGE. The interaction of glycated hemoglobin, serum albumin and type one collagen with P. gingivalis cells or HmuY protein was examined using spectroscopic methods, SDS-PAGE and co-culturing P. gingivalis with human keratinocytes. We found that glycation increases the ability of P. gingivalis to acquire heme from hemoglobin, mostly due to heme sequestration by the HmuY hemophore-like protein. We also found an increase in biofilm formation on glycated collagen-coated abiotic surfaces. We conclude that glycation might promote the virulence of P. gingivalis by making heme more available from hemoglobin and facilitating bacterial biofilm formation, thus increasing P. gingivalis pathogenic potential in vivo.

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