Publications by authors named "Kurt Stenmark"

214 Publications

17β-estradiol and estrogen receptor-α protect right ventricular function in pulmonary hypertension via BMPR2 and apelin.

J Clin Invest 2021 Jan 26. Epub 2021 Jan 26.

Department of Medicine, Indiana University School of Medicine, Indianapolis, United States of America.

Women with pulmonary arterial hypertension (PAH) exhibit better right ventricular (RV) function and survival than men; however, the underlying mechanisms are unknown. We hypothesized that 17β-estradiol (E2), through estrogen receptor α (ERα), attenuates PAH-induced RV failure (RVF) by up-regulating the pro-contractile and pro-survival peptide apelin via a bone morphogenetic protein receptor 2 (BMPR2)-dependent mechanism. We report that ERα and apelin levels are decreased in RV homogenates from patients with RVF and from rats with maladaptive (but not adaptive) RV remodeling. RV cardiomyocyte apelin abundance increased in vivo or in vitro after treatment with E2 or ERα agonist. Studies employing ERα or ERβ null mice, ERα mutant rats or siRNA demonstrated that ERα is necessary for E2 to upregulate RV apelin. E2 and ERα increased BMPR2 in PH-RVs and in isolated RV cardiomyocytes, associated with ERα binding to the Bmpr2 promoter. BMPR2 is required for E2-mediated increases in apelin abundance, and both BMPR2 and apelin are necessary for E2 to enhance pro-survival signaling. E2 or ERα agonist rescued monocrotaline-PH and restored RV apelin and BMPR2 expression. We identified a novel cardioprotective E2-ERα-BMPR2-apelin axis in the RV. Harnessing this axis may lead to novel, RV-targeted therapies for PAH patients of either sex.
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http://dx.doi.org/10.1172/JCI129433DOI Listing
January 2021

Platelet activation contributes to hypoxia-induced inflammation.

Am J Physiol Lung Cell Mol Physiol 2021 Mar 2;320(3):L413-L421. Epub 2020 Dec 2.

Cardiovascular Pulmonary Research Laboratories, University of Colorado, Anschutz Medical Campus, Aurora, Colorado.

Inflammation is central to the pathogenesis of pulmonary vascular remodeling and pulmonary hypertension (PH). Inflammation precedes remodeling in preclinical models, thus supporting the concept that changes in immunity drive remodeling in PH. Platelets are recognized as mediators of inflammation, but whether platelets contribute to hypoxia-driven inflammation has not been studied. We utilized a murine hypoxia model to test the hypothesis that platelets drive hypoxia-induced inflammation. We evaluated male and female 9-wk-old normoxic and hypoxic mice and in selected experiments included hypoxic thrombocytopenic mice. Thrombocytopenic mice were generated with an anti-GP1bα rat IgG antibody. We also performed immunostaining of lung sections from failed donor controls and patients with idiopathic pulmonary arterial hypertension. We found that platelets are increased in the lungs of hypoxic mice and hypoxia induces platelet activation. Platelet depletion prevents hypoxia-driven increases in the proinflammatory chemokines CXCL4 and CCL5 and attenuates hypoxia-induced increase in plasma CSF-2. Pulmonary interstitial macrophages are increased in the lungs of hypoxic mice; this increase is prevented in thrombocytopenic mice. To determine the potential relevance to human disease, lung sections from donors and patients with advanced idiopathic pulmonary arterial hypertension (iPAH) were immunostained for the platelet-specific protein CD41. We observed iPAH lungs had a two-fold increase in CD41, compared with controls. Our data provide evidence that the platelet count is increased in the lungs and activated in mice with hypoxia-induced inflammation and provides rationale for the further study of the potential contribution of platelets to inflammatory mediated vascular remodeling and PH.
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http://dx.doi.org/10.1152/ajplung.00519.2020DOI Listing
March 2021

P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases.

Int J Mol Sci 2020 Sep 18;21(18). Epub 2020 Sep 18.

The Department of Medicine Cardiovascular and Pulmonary Research Laboratory, University of Colorado Denver, Aurora, CO 80045, USA.

Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a "calm" or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a "activated" state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells.
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http://dx.doi.org/10.3390/ijms21186855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555413PMC
September 2020

Pulmonary-arterial-hypertension (PAH)-on-a-chip: fabrication, validation and application.

Lab Chip 2020 09 4;20(18):3334-3345. Epub 2020 Aug 4.

Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Jerry H. Hodge School of Pharmacy, 1300 Coulter Dr., Amarillo, 79119 Texas, USA.

Currently used animal and cellular models for pulmonary arterial hypertension (PAH) only partially recapitulate its pathophysiology in humans and are thus inadequate in reproducing the hallmarks of the disease, inconsistent in portraying the sex-disparity, and unyielding to combinatorial study designs. Here we sought to deploy the ingenuity of microengineering in developing and validating a tissue chip model for human PAH. We designed and fabricated a microfluidic device to emulate the luminal, intimal, medial, adventitial, and perivascular layers of a pulmonary artery. By growing three types of pulmonary arterial cells (PACs)-endothelial, smooth muscle, and adventitial cells, we recreated the PAH pathophysiology on the device. Diseased (PAH) PACs, when grown on the chips, moved of out their designated layers and created phenomena similar to the major pathologies of human PAH: intimal thickening, muscularization, and arterial remodeling and show an endothelial to mesenchymal transition. Flow-induced stress caused control cells, grown on the chips, to undergo morphological changes and elicit arterial remodeling. Our data also suggest that the newly developed chips can be used to elucidate the sex disparity in PAH and to study the therapeutic efficacy of existing and investigational anti-PAH drugs. We believe this miniaturized device can be deployed for testing various prevailing and new hypotheses regarding the pathobiology and drug therapy in human PAH.
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http://dx.doi.org/10.1039/d0lc00605jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7592346PMC
September 2020

The effect of dietary nitrate supplementation on the speed-duration relationship in mice with sickle cell disease.

J Appl Physiol (1985) 2020 09 23;129(3):474-482. Epub 2020 Jul 23.

Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado.

Sickle cell disease (SCD) causes exercise intolerance likely due to impaired skeletal muscle function and low nitric oxide (NO) bioavailability. Dietary nitrate improves hemodynamic and metabolic control during exercise in humans and animals. The purpose of this investigation was to assess the impact of nitrate supplementation on exercise capacity as measured by the running speed to exercise duration relationship [critical speed (CS)]in mice with SCD. We tested the hypothesis that nitrate supplementation via beetroot juice (BR) would attenuate the exercise intolerance observed in mice with SCD. Ten wild-type (WT) and 18 Berkley sickle-cell mice (BERK) received water (WT: = 10, BERK: = 10) or nitrate-rich BR (BERK+BR: = 8, nitrate dose 1 mmol/kg/day) for 5 days. Following the supplementation period, all mice performed 3-5 constant-speed treadmill tests that resulted in exhaustion within 1.5 to 20 min. Time to exhaustion vs. treadmill speed was fit to a hyperbolic model to determine CS. CS was significantly lower in BERK vs. WT and BERK+BR with no significant difference between WT and BERK+BR (WT: 36.6 ± 1.6, BERK: 23.8 ± 1.5, BERK+BR: 31.1 ± 2.1 m/min, < 0.05). Exercise tolerance, measured via CS, was significantly lower in BERK mice relative to WT. However, BERK mice receiving 5 days of nitrate supplementation exhibited no difference in exercise tolerance when compared with WT. These results support the potential utility of a dietary nitrate intervention to improve functionality in SCD patients. Sickle cell disease compromises muscle O delivery resulting in exercise intolerance. Dietary nitrate supplementation increases skeletal muscle blood flow during exercise and may improve exercise capacity in a mouse model of sickle cell disease. We investigated the effects of dietary nitrate supplementation on exercise tolerance in a mouse model of sickle cell disease using the treadmill speed-duration relationship (critical speed). Mice with sickle cell disease provided with a dietary nitrate supplement had a critical speed not significantly different from healthy wild-type mice.
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http://dx.doi.org/10.1152/japplphysiol.00122.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7517430PMC
September 2020

Inflammation, immunity, and vascular remodeling in pulmonary hypertension; Evidence for complement involvement?

Glob Cardiol Sci Pract 2020 Apr 30;2020(1):e202001. Epub 2020 Apr 30.

University of Colorado, Denver, Anschutz Medical Campus, USA.

Pulmonary (arterial) hypertension (PH/PAH) is a life-threatening cardiopulmonary disorder. Experimental evidence suggests involvement of inflammatory and autoimmune processes in pathogenesis of PH/PAH, however the triggering and disease-promoting mechanisms remain unknown. The complement system is a key arm of innate immunity implicated in various pro-inflammatory and autoimmune diseases, yet, surprisingly little is known about the role of complement in PH/PAH pathogenesis. The preponderance of the existing data associates complement with PH/PAH via analysis of plasma and does not study the lung directly. Therefore, we aimed to resolve this by analyzing both the mechanisms of local lung-specific complement activation and the correlation of dysregulated plasma complement to clinical outcome in PAH patients. In our recent studies, reviewed herein, we show, for the first time, that  immunoglobulin-driven activation of the complement cascade, specifically its alternative pathway, in the pulmonary perivascular areas, is a key mechanism initiating pro-inflammatory processes in the early stage of experimental hypoxic PH (a form of "sterile inflammation"). In human patients with end-stage PAH, we have demonstrated that perivascular deposition of immunoglobulin G (IgG) and activation of the complement cascade are "longitudinally" persistent in the disease. We also showed, using unbiased network analysis, that plasma complement signaling, including again the Alternative pathway, is a prognostic factor of survival in patients with idiopathic PAH (IPAH). Based on these initial findings, we suggest that vascular-specific, immunoglobulin-driven dysregulated complement signaling triggers and maintains pulmonary vascular remodeling and PH. Future experiments in this area would facilitate discoveries on whether complement signaling can serve both as a biomarker and therapeutic target in PH/PAH.
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http://dx.doi.org/10.21542/gcsp.2020.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232865PMC
April 2020

RNA-Binding Proteins in Pulmonary Hypertension.

Int J Mol Sci 2020 May 26;21(11). Epub 2020 May 26.

Cardiovascular Pulmonary Research Laboratories, Departments of Pediatrics and Medicine, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.

Pulmonary hypertension (PH) is a life-threatening disease characterized by significant vascular remodeling and aberrant expression of genes involved in inflammation, apoptosis resistance, proliferation, and metabolism. Effective therapeutic strategies are limited, as mechanisms underlying PH pathophysiology, especially abnormal expression of genes, remain unclear. Most PH studies on gene expression have focused on gene transcription. However, post-transcriptional alterations have been shown to play a critical role in inflammation and metabolic changes in diseases such as cancer and systemic cardiovascular diseases. In these diseases, RNA-binding proteins (RBPs) have been recognized as important regulators of aberrant gene expression via post-transcriptional regulation; however, their role in PH is less clear. Identifying RBPs in PH is of great importance to better understand PH pathophysiology and to identify new targets for PH treatment. In this manuscript, we review the current knowledge on the role of dysregulated RBPs in abnormal mRNA gene expression as well as aberrant non-coding RNA processing and expression (e.g., miRNAs) in PH.
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http://dx.doi.org/10.3390/ijms21113757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312837PMC
May 2020

Extracellular adenosine enhances pulmonary artery vasa vasorum endothelial cell barrier function via Gi/ELMO1/Rac1/PKA-dependent signaling mechanisms.

Am J Physiol Cell Physiol 2020 07 20;319(1):C183-C193. Epub 2020 May 20.

Department of Pediatrics, University of Colorado Denver, Aurora, Colorado.

The vasa vasorum (VV), the microvascular network around large vessels, has been recognized as an important contributor to the pathological vascular remodeling in cardiovascular diseases. In bovine and rat models of hypoxic pulmonary hypertension (PH), we have previously shown that chronic hypoxia profoundly increased pulmonary artery (PA) VV permeability, associated with infiltration of inflammatory and progenitor cells in the arterial wall, perivascular inflammation, and structural vascular remodeling. Extracellular adenosine was shown to exhibit a barrier-protective effect on VV endothelial cells (VVEC) via cAMP-independent mechanisms, which involved adenosine A1 receptor-mediated activation of Gi-phosphoinositide 3-kinase-Akt pathway and actin cytoskeleton remodeling. Using VVEC isolated from the adventitia of calf PA, in this study we investigated in more detail the mechanisms linking Gi activation to downstream barrier protection pathways. Using a small-interference RNA (siRNA) technique and transendothelial electrical resistance assay, we found that the adaptor protein, engulfment and cell motility 1 (ELMO1), the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2, and atypical Gi- and Rac1-mediated protein kinase A activation are implicated in VVEC barrier enhancement. In contrast, the actin-interacting GTP-binding protein, girdin, and the p21-activated kinase 1 downstream target, LIM kinase, are not involved in this response. In addition, adenosine-dependent cytoskeletal rearrangement involves activation of cofilin and inactivation of ezrin-radixin-moesin regulatory cytoskeletal proteins, consistent with a barrier-protective mechanism. Collectively, our data indicate that targeting adenosine receptors and downstream barrier-protective pathways in VVEC may have a potential translational significance in developing pharmacological approach for the VV barrier protection in PH.
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http://dx.doi.org/10.1152/ajpcell.00505.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468889PMC
July 2020

Clickable decellularized extracellular matrix as a new tool for building hybrid-hydrogels to model chronic fibrotic diseases in vitro.

J Mater Chem B 2020 08 28;8(31):6814-6826. Epub 2020 Apr 28.

Department of Bioengineering, University of Colorado, Anschutz Medical Campus, 12700 E 19th Ave MS C272 Aurora, Denver, CO 80045, USA.

Fibrotic disorders account for over one third of mortalities worldwide. Despite great efforts to study the cellular and molecular processes underlying fibrosis, there are currently few effective therapies. Dual-stage polymerization reactions are an innovative tool for recreating heterogeneous increases in extracellular matrix (ECM) modulus, a hallmark of fibrotic diseases in vivo. Here, we present a clickable decellularized ECM (dECM) crosslinker incorporated into a dynamically responsive poly(ethylene glycol)-α-methacrylate (PEGαMA) hybrid-hydrogel to recreate ECM remodeling in vitro. An off-stoichiometry thiol-ene Michael addition between PEGαMA (8-arm, 10 kg mol) and the clickable dECM resulted in hydrogels with an elastic modulus of E = 3.6 ± 0.24 kPa, approximating healthy lung tissue (1-5 kPa). Next, residual αMA groups were reacted via a photo-initiated homopolymerization to increase modulus values to fibrotic levels (E = 13.4 ± 0.82 kPa) in situ. Hydrogels with increased elastic moduli, mimicking fibrotic ECM, induced a significant increase in the expression of myofibroblast transgenes. The proportion of primary fibroblasts from dual-reporter mouse lungs expressing collagen 1a1 and alpha-smooth muscle actin increased by approximately 60% when cultured on stiff and dynamically stiffened hybrid-hydrogels compared to soft. Likewise, fibroblasts expressed significantly increased levels of the collagen 1a1 transgene on stiff regions of spatially patterned hybrid-hydrogels compared to the soft areas. Collectively, these results indicate that hybrid-hydrogels are a new tool that can be implemented to spatiotemporally induce a phenotypic transition in primary murine fibroblasts in vitro.
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http://dx.doi.org/10.1039/d0tb00613kDOI Listing
August 2020

Band on the run: insights into right ventricular reverse remodelling.

Cardiovasc Res 2020 Aug;116(10):1651-1653

Cardiovascular Pulmonary Research Laboratories, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA.

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http://dx.doi.org/10.1093/cvr/cvaa091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380720PMC
August 2020

Perspective: pathobiological paradigms in pulmonary hypertension, time for reappraisal.

Am J Physiol Lung Cell Mol Physiol 2020 06 18;318(6):L1131-L1137. Epub 2020 Mar 18.

Cardiovascular Pulmonary Research Laboratories, Division of Critical Care Medicine, Departments of Pediatrics and Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado.

For the past 120 years, there has been a progressive evolution of the pathobiological concepts underlying pulmonary hypertension. Conceptual frameworks, build around the paradigms of excessive vasoconstriction (vs. vasodilation) and, more recently, of the cancer-like hypothesis of pulmonary hypertension, have served to consolidate key discoveries; moreover, they have and continue contributing to innovative advances that have been translated into either successful or potential new therapies. However, those frameworks do not fully address the complexity and challenges facing pulmonary hypertension, particularly those involving the marked heterogeneity of disease presentation and the dynamic changes occurring over time in affected tissues and cells. This is particularly relevant in regards to the molecular pathways of pulmonary hypertension; the ever growing understanding of molecular and cellular pathways requires clarification if they drive distinctive pulmonary vascular lesions in a given lung and disease patients with the same group pulmonary hypertension. Novel methodologies and approaches can start dissecting this key challenge in the field as it is critical to address the key angle of heterogeneity of the disease and reappraisal of disease-modifying therapies.
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http://dx.doi.org/10.1152/ajplung.00067.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347272PMC
June 2020

Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.

Am J Physiol Lung Cell Mol Physiol 2020 04 11;318(4):L773-L786. Epub 2020 Mar 11.

Department of Pharmacology, New York Medical College, Valhalla, New York.

Metabolic reprogramming is considered important in the pathogenesis of the occlusive vasculopathy observed in pulmonary hypertension (PH). However, the mechanisms that link reprogrammed metabolism to aberrant expression of genes, which modulate functional phenotypes of cells in PH, remain enigmatic. Herein, we demonstrate that, in mice, hypoxia-induced PH was prevented by glucose-6-phosphate dehydrogenase deficiency (G6PD), and further show that established severe PH in mice was attenuated by knockdown with G6PD shRNA or by G6PD inhibition with an inhibitor (N-ethyl-N'-[(3β,5α)-17-oxoandrostan-3-yl]urea, NEOU). Mechanistically, G6PD, knockdown and inhibition in lungs: ) reduced hypoxia-induced changes in cytoplasmic and mitochondrial metabolism, ) increased expression of Tet methylcytosine dioxygenase 2 () gene, and ) upregulated expression of the coding genes and long noncoding (lnc) RNA , which inhibits cell growth, by hypomethylating the promoter flanking region downstream of the transcription start site. These results suggest functional TET2 is required for G6PD inhibition to increase gene expression and to reverse hypoxia-induced PH in mice. Furthermore, the inhibitor of G6PD activity (NEOU) decreased metabolic reprogramming, upregulated , and inhibited growth of control and diseased smooth muscle cells isolated from pulmonary arteries of normal individuals and idiopathic-PAH patients, respectively. Collectively, these findings demonstrate a previously unrecognized function for G6PD as a regulator of DNA methylation. These findings further suggest that G6PD acts as a link between reprogrammed metabolism and aberrant gene regulation and plays a crucial role in regulating the phenotype of cells implicated in the pathogenesis of PH, a debilitating disorder with a high mortality rate.
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http://dx.doi.org/10.1152/ajplung.00001.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191486PMC
April 2020

Publisher Correction: Stable isotope metabolomics of pulmonary artery smooth muscle and endothelial cells in pulmonary hypertension and with TGF-beta treatment.

Sci Rep 2020 Mar 4;10(1):4349. Epub 2020 Mar 4.

Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-60500-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055208PMC
March 2020

c-Jun, Foxo3a, and c-Myc Transcription Factors are Key Regulators of ATP-Mediated Angiogenic Responses in Pulmonary Artery Vasa Vasorum Endothelial Cells.

Cells 2020 02 11;9(2). Epub 2020 Feb 11.

University of Colorado Denver, Department of Medicine Cardiovascular and Pulmonary Research Laboratory, Aurora, CO 80045, USA.

Angiogenic vasa vasorum (VV) expansion plays an essential role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH), a cardiovascular disease. We previously showed that extracellular ATP released under hypoxic conditions is an autocrine/paracrine, the angiogenic factor for pulmonary artery (PA) VV endothelial cells (VVECs), acting via P2Y purinergic receptors (P2YR) and the Phosphoinositide 3-kinase (PI3K)-Akt-Mammalian Target of Rapamycin (mTOR) signaling. To further elucidate the molecular mechanisms of ATP-mediated VV angiogenesis, we determined the profile of ATP-inducible transcription factors (TFs) in VVECs using a TranSignal protein/DNA array. C-Jun, c-Myc, and Foxo3 were found to be upregulated in most VVEC populations and formed nodes connecting several signaling networks. siRNA-mediated knockdown (KD) of these TFs revealed their critical role in ATP-induced VVEC angiogenic responses and the regulation of downstream targets involved in tissue remodeling, cell cycle control, expression of endothelial markers, cell adhesion, and junction proteins. Our results showed that c-Jun was required for the expression of ATP-stimulated angiogenic genes, c-Myc was repressive to anti-angiogenic genes, and Foxo3a predominantly controlled the expression of anti-apoptotic and junctional proteins. The findings from our study suggest that pharmacological targeting of the components of P2YR-PI3K-Akt-mTOR axis and specific TFs reduced ATP-mediated VVEC angiogenic response and may have a potential translational significance in attenuating pathological vascular remodeling.
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http://dx.doi.org/10.3390/cells9020416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072142PMC
February 2020

Stable isotope metabolomics of pulmonary artery smooth muscle and endothelial cells in pulmonary hypertension and with TGF-beta treatment.

Sci Rep 2020 01 15;10(1):413. Epub 2020 Jan 15.

Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Altered metabolism in pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs) contributes to the pathology of pulmonary hypertension (PH), but changes in substrate uptake and how substrates are utilized have not been fully characterized. We hypothesized stable isotope metabolomics would identify increased glucose, glutamine and fatty acid uptake and utilization in human PASMCs and PAECs from PH versus control specimens, and that TGF-β treatment would phenocopy these metabolic changes. We used C-labeled glucose, glutamine or a long-chain fatty acid mixture added to cell culture media, and mass spectrometry-based metabolomics to detect and quantify C-labeled metabolites. We found PH PASMCs had increased glucose uptake and utilization by glycolysis and the pentose shunt, but no changes in glutamine or fatty acid uptake or utilization. Diseased PAECs had increased proximate glycolysis pathway intermediates, less pentose shunt flux, increased anaplerosis from glutamine, and decreased fatty acid β-oxidation. TGF-β treatment increased glycolysis in PASMCs, but did not recapitulate the PAEC disease phenotype. In TGF-β-treated PASMCs, glucose, glutamine and fatty acids all contributed carbons to the TCA cycle. In conclusion, PASMCs and PAECs collected from PH subjects have significant changes in metabolite uptake and utilization, partially recapitulated by TGF-β treatment.
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http://dx.doi.org/10.1038/s41598-019-57200-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962446PMC
January 2020

Pluripotent hematopoietic stem cells augment α-adrenergic receptor-mediated contraction of pulmonary artery and contribute to the pathogenesis of pulmonary hypertension.

Am J Physiol Lung Cell Mol Physiol 2020 02 8;318(2):L386-L401. Epub 2020 Jan 8.

Department of Pharmacology, New York Medical College, Valhalla, New York.

Pulmonary hypertension (PH) is a multicellular and progressive disease with a high mortality rate. Among many cell types, hematopoietic stem cells (HSCs) are incriminated in the pathogenesis of PH. However, our understanding of the mechanisms that increase HSCs in blood and lungs of hypertensive animals or patients and the role played by HSCs in the pathogenesis of PH remains elusive. Studies suggest that glycolysis is critical for the survival and growth of HSCs. In various cell types from hypertensive lungs of animals and patients, glycolysis and the glucose-6-phosphate dehydrogenase (G6PD) activity are increased. Herein, we demonstrated in mice that chronic hypoxia increased HSCs (CD34, CD117, CD133, CD34/CD117, and CD34/CD133) in bone marrow and blood and around hypertensive pulmonary arteries in a time-dependent manner. Intriguingly, we found fewer CD133 cells in the bone marrow of C57BL/6 mice compared with Sv129J mice, and C57BL mice developed less severe chronic hypoxia-elicited PH and heart failure than Sv129J mice. Similarly, the numbers of CD34 and CD117 cells in blood of patients with pulmonary arterial hypertension (PAH) were higher (>3-fold) compared with healthy individuals. By allogeneic bone marrow transplantation, we found that GFP bone marrow cells infiltrated the lungs and accumulated around the pulmonary arteries in lungs of hypoxic mice, and these cells contributed to increased α-adrenergic receptor-mediated contraction of the pulmonary artery cultured in hypoxia. Inhibition of G6PD activity with (3β,5α)-3,21-dihydroxypregnan-20-one, a novel and potent G6PD inhibitor, decreased HSCs in bone marrow, blood, and lungs of hypoxic mice and reduced α-agonist-induced contraction of the pulmonary artery and established hypoxia-induced PH. We did not observe CD133 cells around the pulmonary arteries in the lungs of chronically hypoxic G6PD-deficient mice. Furthermore, knockdown of G6PD and inhibition of G6PD activity: ) downregulated canonical and noncanonical and receptors genes; ) upregulated ; ) decreased , and ) reduced HSC (CD117 and CD133) numbers. In all, our findings demonstrate unexpected function for bone marrow-derived HSCs in augmenting α-adrenergic receptor-mediated contraction of pulmonary arteries and remodeling of pulmonary arteries that contribute to increase pulmonary vascular resistance in PAH patients and hypoxic mice and suggest that G6PD, by regulating expression of genes in the WNT and BMPR signaling, contributed to increase and release of HSCs from the bone marrow in response to hypoxic stimuli.
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http://dx.doi.org/10.1152/ajplung.00327.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052680PMC
February 2020

Hot topics in the mechanisms of pulmonary arterial hypertension disease: cancer-like pathobiology, the role of the adventitia, systemic involvement, and right ventricular failure.

Pulm Circ 2019 Oct-Dec;9(4):2045894019889775. Epub 2019 Nov 20.

Institute of Physiology, Charité - Universitaetsmedizin Berlin, Berlin, Germany.

In order to intervene appropriately and develop disease-modifying therapeutics for pulmonary arterial hypertension, it is crucial to understand the mechanisms of disease pathogenesis and progression. We herein discuss four topics of disease mechanisms that are currently highly debated, yet still unsolved, in the field of pulmonary arterial hypertension. Is pulmonary arterial hypertension a cancer-like disease? Does the adventitia play an important role in the initiation of pulmonary vascular remodeling? Is pulmonary arterial hypertension a systemic disease? Does capillary loss drive right ventricular failure? While pulmonary arterial hypertension does not replicate all features of cancer, anti-proliferative cancer therapeutics might still be beneficial in pulmonary arterial hypertension if monitored for safety and tolerability. It was recognized that the adventitia as a cell-rich compartment is important in the disease pathogenesis of pulmonary arterial hypertension and should be a therapeutic target, albeit the data are inconclusive as to whether the adventitia is involved in the initiation of neointima formation. There was agreement that systemic diseases can lead to pulmonary arterial hypertension and that pulmonary arterial hypertension can have systemic effects related to the advanced lung pathology, yet there was less agreement on whether idiopathic pulmonary arterial hypertension is a systemic disease per se. Despite acknowledging the limitations of exactly assessing vascular density in the right ventricle, it was recognized that the failing right ventricle may show inadequate vascular adaptation resulting in inadequate delivery of oxygen and other metabolites. Although the debate was not meant to result in a definite resolution of the specific arguments, it sparked ideas about how we might resolve the discrepancies by improving our disease modeling (rodent models, large-animal studies, studies of human cells, tissues, and organs) as well as standardization of the models. Novel experimental approaches, such as lineage tracing and better three-dimensional imaging of experimental as well as human lung and heart tissues, might unravel how different cells contribute to the disease pathology.
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http://dx.doi.org/10.1177/2045894019889775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868582PMC
November 2019

Targeting histone acetylation in pulmonary hypertension and right ventricular hypertrophy.

Br J Pharmacol 2021 Jan 26;178(1):54-71. Epub 2020 Jan 26.

Department of Lung Development and Remodeling, Max Planck Institute for Heart and Lung Research, Member of the German Center for Lung Research (DZL), Member of the Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany.

Epigenetic mechanisms, including DNA methylation and histone post-translational modifications (PTMs), have been known to regulate chromatin structure and lineage-specific gene expression during cardiovascular development and disease. However, alterations in the landscape of histone PTMs and their contribution to the pathogenesis of incurable cardiovascular diseases such as pulmonary hypertension (PH) and associated right heart failure (RHF) remain largely unexplored. This review focusses on the studies in PH and RHF that investigated the gene families that write (histone acetyltransferases), read (bromodomain-containing proteins) or erase (histone deacetylases [HDACs] and sirtuins [SIRT]) acetyl moieties from the ε-amino group of lysine residues of histones and non-histone proteins. Analysis of cells and tissues isolated from the in vivo preclinical models of PH and human pulmonary arterial hypertension not only confirmed significant alterations in the expression levels of multiple HDACs, SIRT1, SIRT3 and BRD4 proteins but also demonstrated their strong association to proliferative, inflammatory and fibrotic phenotypes linked to the pathological vascular remodelling process. Due to the reversible nature of post-translational protein acetylation, the therapeutic efficacy of numerous small-molecule inhibitors (vorinostat, valproic acid, sodium butyrate, mocetinostat, entinostat, tubastatin A, apabetalone, JQ1 and resveratrol) have been evaluated in different preclinical models of cardiovascular disease, which revealed the promising therapeutic benefits of targeting histone acetylation pathways in the attenuation of cardiac hypertrophy, fibrosis, left heart dysfunction, PH and RHF. This review also emphasizes the need for deeper molecular insights into the contribution of epigenetic changes to PH pathogenesis and therapeutic evaluation of isoform-specific modulation in ex vivo and in vivo models of PH and RHF. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc.
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http://dx.doi.org/10.1111/bph.14932DOI Listing
January 2021

A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension.

Nat Commun 2019 11 15;10(1):5183. Epub 2019 Nov 15.

Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, S10 2RX, UK.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase life expectancy but have limited impact on the progressive pulmonary vascular remodelling that drives PAH. Osteoprotegerin (OPG) is increased within serum and lesions of patients with idiopathic PAH and is a mitogen and migratory stimulus for pulmonary artery smooth muscle cells (PASMCs). Here, we report that the pro-proliferative and migratory phenotype in PASMCs stimulated with OPG is mediated via the Fas receptor and that treatment with a human antibody targeting OPG can attenuate pulmonary vascular remodelling associated with PAH in multiple rodent models of early and late treatment. We also demonstrate that the therapeutic efficacy of the anti-OPG antibody approach in the presence of standard of care vasodilator therapy is mediated by a reduction in pulmonary vascular remodelling. Targeting OPG with a therapeutic antibody is a potential treatment strategy in PAH.
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http://dx.doi.org/10.1038/s41467-019-13139-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858314PMC
November 2019

Interstitial macrophage-derived thrombospondin-1 contributes to hypoxia-induced pulmonary hypertension.

Cardiovasc Res 2020 Oct;116(12):2021-2030

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Zuckerberg San Francisco General Hospital and Trauma Center, University of California, San Francisco, Building 100, 3rd floor, 1001 Potrero Ave, San Francisco, CA 94110, USA.

Aims: Transforming growth factor-β (TGF-β) signalling is required for chronic hypoxia-induced pulmonary hypertension (PH). The activation of TGF-β by thrombospondin-1 (TSP-1) contributes to the pathogenesis of hypoxia-induced PH. However, neither the cellular source of pathologic TSP-1 nor the downstream signalling pathway that link activated TGF-β to PH have been determined. In this study, we hypothesized that circulating monocytes, which are recruited to become interstitial macrophages (IMs), are the major source of TSP-1 in hypoxia-exposed mice, and TSP-1 activates TGF-β with increased Rho-kinase signalling, causing vasoconstriction.

Methods And Results: Flow cytometry revealed that a specific subset of IMs is the major source of pathologic TSP-1 in hypoxia. Intravenous depletion and parabiosis experiments demonstrated that these cells are circulating prior to recruitment into the interstitium. Rho-kinase-mediated vasoconstriction was a major downstream target of active TGF-β. Thbs1 deficient bone marrow (BM) protected against hypoxic-PH by blocking TGF-β activation and Rho-kinase-mediated vasoconstriction.

Conclusion: In hypoxia-challenged mice, BM derived and circulating monocytes are recruited to become IMs which express TSP-1, resulting in TGF-β activation and Rho-kinase-mediated vasoconstriction.
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http://dx.doi.org/10.1093/cvr/cvz304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519884PMC
October 2020

Role of Inflammatory Cell Subtypes in Heart Failure.

J Immunol Res 2019 2;2019:2164017. Epub 2019 Sep 2.

Cardiovascular Pulmonary Research Laboratory, University of Colorado Denver, Aurora CO, USA.

Inflammation is a well-known feature of heart failure. Studies have shown that while some inflammation is required for repair during injury and is protective, prolonged inflammation leads to myocardial remodeling and apoptosis of cardiac myocytes. Various types of immune cells are implicated in myocardial inflammation and include neutrophils, macrophages, eosinophils, mast cells, natural killer cells, T cells, and B cells. Recent clinical trials have targeted inflammatory cascades as therapy for heart failure with limited success. A better understanding of the temporal course of the infiltration of the different immune cells and their contribution to the inflammatory process may improve the success for therapy. This brief review outlines the major cell types involved in heart failure, and some of their actions are summarized in the supplementary figure.
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http://dx.doi.org/10.1155/2019/2164017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745095PMC
February 2020

Redistribution of EC-SOD resolves bleomycin-induced inflammation increased apoptosis of recruited alveolar macrophages.

FASEB J 2019 12 27;33(12):13465-13475. Epub 2019 Sep 27.

Cardiovascular Pulmonary Research Laboratories, Departments of Pediatrics and Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

A human single nucleotide polymorphism (SNP) in the matrix-binding domain of extracellular superoxide dismutase (EC-SOD), with arginine to glycine substitution at position 213 (R213G), redistributes EC-SOD from the matrix into extracellular fluids. We reported that, following bleomycin (bleo), knockin mice harboring the human R213G SNP (R213G mice) exhibit enhanced resolution of inflammation and protection against fibrosis, compared with wild-type (WT) littermates. In this study, we tested the hypothesis that the EC-SOD R213G SNP promotes resolution accelerated apoptosis of recruited alveolar macrophage (AM). RNA sequencing and Ingenuity Pathway Analysis 7 d postbleo in recruited AM implicated increased apoptosis and blunted inflammatory responses in the R213G strain exhibiting accelerated resolution. We validated that the percentage of apoptosis was significantly elevated in R213G recruited AM WT at 3 and 7 d postbleo . Recruited AM numbers were also significantly decreased in R213G mice WT at 3 and 7 d postbleo. ChaC glutathione-specific γ-glutamylcyclotransferase 1 (Chac1), a proapoptotic γ-glutamyl cyclotransferase that depletes glutathione, was increased in the R213G recruited AM. Overexpression of Chac1 induced apoptosis of macrophages and was blocked by administration of cell-permeable glutathione. In summary, we provide new evidence that redistributed EC-SOD accelerates the resolution of inflammation through redox-regulated mechanisms that increase recruited AM apoptosis.-Allawzi, A., McDermott, I., Delaney, C., Nguyen, K., Banimostafa, L., Trumpie, A., Hernandez-Lagunas, L., Riemondy, K., Gillen, A., Hesselberth, J., El Kasmi, K., Sucharov, C. C., Janssen, W. J., Stenmark, K., Bowler, R., Nozik-Grayck, E. Redistribution of EC-SOD resolves bleomycin-induced inflammation increased apoptosis of recruited alveolar macrophages.
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http://dx.doi.org/10.1096/fj.201901038RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894081PMC
December 2019

Immunoglobulin-driven Complement Activation Regulates Proinflammatory Remodeling in Pulmonary Hypertension.

Am J Respir Crit Care Med 2020 01;201(2):224-239

Division of Critical Care Medicine and Cardiovascular Pulmonary Research, Departments of Pediatrics and Medicine.

Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder in which inflammation and immunity have emerged as critical early pathogenic elements. Although proinflammatory processes in PH and pulmonary arterial hypertension (PAH) are the focus of extensive investigation, the initiating mechanisms remain elusive. We tested whether activation of the complement cascade is critical in regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and can serve as a prognostic biomarker of outcome in human PAH. We used immunostaining of lung tissues from experimental PH models and patients with PAH, analyses of genetic murine models lacking specific complement components or circulating immunoglobulins, cultured human pulmonary adventitial fibroblasts, and network medicine analysis of a biomarker risk panel from plasma of patients with PAH. Pulmonary perivascular-specific activation of the complement cascade was identified as a consistent critical determinant of PH and PAH in experimental animal models and humans. In experimental hypoxic PH, proinflammatory and pro-proliferative responses were dependent on complement (alternative pathway and component 5), and immunoglobulins, particularly IgG, were critical for activation of the complement cascade. We identified Csf2/GM-CSF as a primary complement-dependent inflammatory mediator. Furthermore, using network medicine analysis of a biomarker risk panel from plasma of patients with PAH, we demonstrated that complement signaling can serve as a prognostic factor for clinical outcome in PAH. This study establishes immunoglobulin-driven dysregulated complement activation as a critical pathobiological mechanism regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and demonstrates complement signaling as a critical determinant of clinical outcome in PAH.
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http://dx.doi.org/10.1164/rccm.201903-0591OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961733PMC
January 2020

Suppression of HIF2 signalling attenuates the initiation of hypoxia-induced pulmonary hypertension.

Eur Respir J 2019 12 12;54(6). Epub 2019 Dec 12.

Cardiovascular Pulmonary Research Laboratories, Division of Pulmonary Sciences and Critical Care Medicine, Division of Pediatrics-Critical Care, Depts of Medicine and Pediatrics, University of Colorado, Aurora, CO, USA

Most published studies addressing the role of hypoxia inducible factors (HIFs) in hypoxia-induced pulmonary hypertension development employ models that may not recapitulate the clinical setting, including the use of animals with pre-existing lung/vascular defects secondary to embryonic HIF ablation or activation. Furthermore, critical questions including how and when HIF signalling contributes to hypoxia-induced pulmonary hypertension remain unanswered.Normal adult rodents in which global HIF1 or HIF2 was inhibited by inducible gene deletion or pharmacological inhibition (antisense oligonucleotides (ASO) and small molecule inhibitors) were exposed to short-term (4 days) or chronic (4-5 weeks) hypoxia. Haemodynamic studies were performed, the animals euthanised, and lungs and hearts obtained for pathological and transcriptomic analysis. Cell-type-specific HIF signals for pulmonary hypertension initiation were determined in normal pulmonary vascular cells and in mice (using cell-type-specific HIF deletion).Global deletion in mice did not prevent hypoxia-induced pulmonary hypertension at 5 weeks. Mice with global deletion did not survive long-term hypoxia. Partial deletion or -ASO (but not -ASO) reduced vessel muscularisation, increases in pulmonary arterial pressures and right ventricular hypertrophy in mice exposed to 4-5 weeks of hypoxia. A small molecule HIF2 inhibitor (PT2567) significantly attenuated early events (monocyte recruitment and vascular cell proliferation) in rats exposed to 4 days of hypoxia, as well as vessel muscularisation, tenascin C accumulation and pulmonary hypertension development in rats exposed to 5 weeks of hypoxia. , HIF2 induced a distinct set of genes in normal human pulmonary vascular endothelial cells, mediating inflammation and proliferation of endothelial cells and smooth muscle cells. Endothelial knockout prevented hypoxia-induced pulmonary hypertension in mice.Inhibition of HIF2 (but not HIF1) can provide a therapeutic approach to prevent the development of hypoxia-induced pulmonary hypertension. Future studies are needed to investigate the role of HIFs in pulmonary hypertension progression and reversal.
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http://dx.doi.org/10.1183/13993003.00378-2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911916PMC
December 2019

Pegloticase and lowering blood pressure in refractory gout; is it uric acid or hydrogen peroxide?

Eur J Intern Med 2019 Nov 27;69:e11-e12. Epub 2019 Aug 27.

Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, Anschutz Medical Campus, University of Colorado-Denver School of Medicine, Aurora, CO, USA; Division of Pulmonary and Critical Care, Department of Pediatrics, Anschutz Medical Campus, University of Colorado-Denver School of Medicine, Aurora, CO, USA.

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http://dx.doi.org/10.1016/j.ejim.2019.08.017DOI Listing
November 2019

An Hb-mediated circulating macrophage contributing to pulmonary vascular remodeling in sickle cell disease.

JCI Insight 2019 08 8;4(15). Epub 2019 Aug 8.

Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, Anschutz Medical Campus, University of Colorado-Denver School of Medicine, Aurora, Colorado, USA.

Circulating macrophages recruited to the lung contribute to pulmonary vascular remodeling in various forms of pulmonary hypertension (PH). In this study we investigated a macrophage phenotype characterized by intracellular iron accumulation and expression of antioxidant (HO-1), vasoactive (ET-1), and proinflammatory (IL-6) mediators observed in the lung tissue of deceased sickle cell disease (SCD) patients with diagnosed PH. To this end, we evaluated an established rat model of group 5 PH that is simultaneously exposed to free hemoglobin (Hb) and hypobaric hypoxia (HX). Here, we tested the hypothesis that pulmonary vascular remodeling observed in human SCD with concomitant PH could be replicated and mechanistically driven in our rat model by a similar macrophage phenotype with iron accumulation and expression of a similar mixture of antioxidant (HO-1), vasoactive (ET-1), and inflammatory (IL-6) proteins. Our data suggest phenotypic similarities between pulmonary perivascular macrophages in our rat model and human SCD with PH, indicating a potentially novel maladaptive immune response to concomitant bouts of Hb and HX exposure. Moreover, by knocking out circulating macrophages with gadolinium trichloride (GdCl3), the response to combined Hb and hypobaric HX was significantly attenuated in rats, suggesting a critical role for macrophages in the exacerbation of SCD PH.
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http://dx.doi.org/10.1172/jci.insight.127860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693838PMC
August 2019

The right ventricular fibroblast secretome drives cardiomyocyte dedifferentiation.

PLoS One 2019 2;14(8):e0220573. Epub 2019 Aug 2.

University of Colorado-Denver, Dept. of Medicine/Cardiology, Aurora, CO, United States of America.

Rationale: In virtually all models of heart failure, prognosis is determined by right ventricular (RV) function; thus, understanding the cellular mechanisms contributing to RV dysfunction is critical. Whole organ remodeling is associated with cell-specific changes, including cardiomyocyte dedifferentiation and activation of cardiac fibroblasts (Cfib) which in turn is linked to disorganization of cytoskeletal proteins and loss of sarcomeric structures. However, how these cellular changes contribute to RV function remains unknown. We've previously shown significant organ-level RV dysfunction in a large animal model of pulmonary hypertension (PH) which was not mirrored by reduced function of isolated cardiomyocytes. We hypothesized that factors produced by the endogenous Cfib contribute to global RV dysfunction by generating a heterogeneous cellular environment populated by dedifferentiated cells.

Objective: To determine the effect of Cfib conditioned media (CM) from the PH calf (PH-CM) on adult rat ventricular myocytes (ARVM) in culture.

Methods And Results: Brief exposure (<2 days) to PH-CM results in rapid, marked dedifferentiation of ARVM to a neonatal-like phenotype exhibiting spontaneous contractile behavior. Dedifferentiated cells maintain viability for over 30 days with continued expression of cardiomyocyte proteins including TnI and α-actinin yet exhibit myofibroblast characteristics including expression of α-smooth muscle actin. Using a bioinformatics approach to identify factor(s) that contribute to dedifferentiation, we found activation of the PH Cfib results in a unique transcriptome correlating with factors both in the secretome and with activated pathways in the dedifferentiated myocyte. Further, we identified upregulation of periostin in the Cfib and CM, and demonstrate that periostin is sufficient to drive cardiomyocyte dedifferentiation.

Conclusions: These data suggest that paracrine factor(s) released by Cfib from the PH calf signal a phenotypic transformation in a population of cardiomyocytes that likely contributes to RV dysfunction. Therapies targeting this process, such as inhibition of periostin, have the potential to prevent RV dysfunction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220573PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677314PMC
March 2020

CAR, a Homing Peptide, Prolongs Pulmonary Preferential Vasodilation by Increasing Pulmonary Retention and Reducing Systemic Absorption of Liposomal Fasudil.

Mol Pharm 2019 08 27;16(8):3414-3429. Epub 2019 Jun 27.

Department of Pharmaceutical Sciences, School of Pharmacy , Texas Tech University Health Sciences Center , Amarillo , Texas 79430 , United States.

Here, we sought to elucidate the role of CAR (a cyclic peptide) in the accumulation and distribution of fasudil, a drug for pulmonary arterial hypertension (PAH), in rat lungs and in producing pulmonary specific vasodilation in PAH rats. As such, we prepared liposomes of fasudil and CAR-conjugated liposomal fasudil and assessed the liposomes for CAR conjugation, physical properties, entrapment efficiencies, in vitro release profiles, and stabilities upon incubation in cell culture media, storage, and aerosolization. We also studied the cellular uptake of fasudil in different formulations, quantified heparan sulfate (HS) in pulmonary arterial smooth muscle cells (PASMCs), and investigated the distribution of the liposomes in the lungs of PAH rats. We assessed the drug accumulation in a close and recirculating isolated perfused rat lung model and studied the pharmacokinetics and pharmacological efficacy of the drug and formulations in Sugen/hypoxia-induced PAH rats. The entrapment efficiency of the liposomal fasudil was 95.5 ± 4.5%, and the cumulative release was 93.95 ± 6.22%. The uptake of CAR liposomes by pulmonary arterial cells and their distribution and accumulation in the lungs were much greater than those of no-CAR-liposomes. CAR-induced increase in the cellular uptake was associated with an increase in HS expression by rat PAH-PASMCs. CAR, when conjugated with liposomal fasudil and given via an intratracheal instillation, extended the elimination half-life of the drug by four-fold compared with fasudil-in-no-CAR-liposomes given via the same route. CAR-conjugated liposomal fasudil, as opposed to fasudil-in-no-CAR-liposomes and CAR pretreatment followed by fasudil-in-no-CAR-liposomes, reduced the mean pulmonary arterial pressure by 40-50% for 6 h, without affecting the mean systemic arterial pressure. On the whole, this study suggests that CAR aids in concentrating the drug in the lungs, increasing the cellular uptake, extending the half-life of fasudil, and eliciting a pulmonary-specific vasodilation when the peptide remains conjugated on the liposomal surface, but not when CAR is given as a pretreatment or alone as an admixture with the drug.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035787PMC
August 2019