Cellular senescence in the pathogenesis of pulmonary arterial hypertension: the good, the bad and the uncertain.

Senescence refers to a cellular state marked by irreversible cell cycle arrest and the secretion of pro-inflammatory and tissue-remodeling factors. The senescence associated secretory phenotype (SASP) impacts the tissue microenvironment and provides cues for the immune system to eliminate senescent cells (SCs). Cellular senescence has a dual nature; it can be beneficial during embryonic development, tissue repair, and tumor suppression, but it can also be detrimental in the context of chronic stress, persistent tissue injury, together with an impairment in SC clearance. Recently, the accumulation of SCs has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), a progressive condition affecting the pre-capillary pulmonary arterial bed. PAH is characterized by endothelial cell (EC) injury, inflammation, and proliferative arterial remodeling, which leads to right heart failure and premature mortality. While vasodilator therapies can improve symptoms, there are currently no approved treatments capable of reversing the obliterative arterial remodeling. Ongoing endothelial injury and dysfunction is central to the development of PAH, perpetuated by hemodynamic perturbation leading to pathological intimal shear stress. The precise role of senescent ECs in PAH remains unclear. Cellular senescence may facilitate endothelial repair, particularly in the early stages of disease. However, in more advanced disease the accumulation of senescent ECs may promote vascular inflammation and occlusive arterial remodeling. In this review, we will examine the evidence that supports a role of endothelial cell senescence to the pathogenesis of PAH. Furthermore, we will compare and discuss the apparent contradictory outcomes with the use of interventions targeting cellular senescence in the context of experimental models of pulmonary hypertension. Finally, we will attempt to propose a framework for the understanding of the complex interplay between EC injury, senescence, inflammation and arterial remodeling, which can guide further research in this area and the development of effective therapeutic strategies.

Key quality parameter comparison of mesenchymal stem cell product cryopreserved in different cryopreservation solutions for clinical applications.

Introduction: Cryopreservation is a critical process of cell products for achieving a commercial viability through wide scale adoption. By preserving cells in a lower temperature, cryopreservation enables a product to be off-the-shelf and ready for infusion. An optimized cryopreservation strategy can maintain the viability, phenotype, and potency of thawed mesenchymal stromal/stem cells (MSCs) while being regulatory compliant. We compared three clinical-ready formulations with one research cryopreservation solutions and evaluated key quality parameters of post thawed MSCs. Method and result: MSCs were cryopreserved at 3, 6, and 9 million cells/mL (M/mL) in four different cryopreservation solutions: NutriFreez (10% dimethyl sulfoxide [DMSO]), Plasmalyte A (PLA)/5% human albumin (HA)/10% DMSO (PHD10), CryoStor CS5 (5% DMSO), and CryoStor CS10 (10% DMSO). To establish post thaw viability, cells were evaluated with no dilution of DMSO (from 3 M/mL), 1:1 dilution (from 6 M/mL), or 1:2 dilution (from 9 M/mL) with PLA/5% HA, to achieve uniform concentration at 3 M/mL. Cell viability was measured at 0-, 2-, 4-, and 6-h post thaw with Trypan blue exclusion and Annexin V/PI staining. Dilution (1:2) of final cell products from 9M/mL resulted in an improvement of cell viability over 6 h but showed a trend of decreased recovery. MSCs cryopreserved in solutions with 10% DMSO displayed comparable viabilities and recoveries up to 6 h after thawing, whereas a decreasing trend was noted in cell viability and recovery with CS5. Cells from all groups exhibited surface marker characteristics of MSCs. We further evaluated cell proliferation after 6-day recovery in culture. While cells cryopreserved in NutriFreez and PHD10 presented similar cell growth post thaw, MSCs cryopreserved in CS5 and CS10 at 3 M/mL and 6M/mL showed 10-fold less proliferative capacity. No significant differences were observed between MSCs cryopreserved in NutriFreez and PHD10 in their potency to inhibit T cell proliferation and improve monocytic phagocytosis. Conclusion: MSCs can be cryopreserved up to 9 M/mL without losing notable viability and recovery, while exhibiting comparable post thaw potency with NutriFreez and PHD10. These results highlight the importance of key parameter testing for selecting the optimal cryopreservation solution for MSC-based therapy.

Single-Cell RNA Sequencing Reveals Repair Features of Human Umbilical Cord Mesenchymal Stromal Cells.

Rationale: The chronic lung disease bronchopulmonary dysplasia (BPD) is the most severe complication of extreme prematurity. BPD results in impaired lung alveolar and vascular development and long-term respiratory morbidity, for which only supportive therapies exist. Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) improve lung structure and function in experimental BPD. Results of clinical trials with MSCs for many disorders do not yet match the promising preclinical studies. A lack of specific criteria to define functionally distinct MSCs persists. Objectives: To determine and correlate single-cell UC-MSC transcriptomic profiles with therapeutic potential. Methods: UC-MSCs from five term donors and human neonatal dermal fibroblasts (HNDFs; control cells of mesenchymal origin) transcriptomes were investigated using single-cell RNA sequencing (scRNA-seq) analysis. The lung-protective effect of UC-MSCs with a distinct transcriptome and control HNDFs was tested in vivo in hyperoxia-induced neonatal lung injury in rats. Measurements and Main Results: UC-MSCs showed limited transcriptomic heterogeneity but were different from HNDFs. Gene Ontology enrichment analysis revealed distinct (progenitor-like and fibroblast-like) UC-MSC subpopulations. Only treatment with progenitor-like UC-MSCs improved lung function and structure and attenuated pulmonary hypertension in hyperoxia-exposed rat pups. Moreover, scRNA-seq identified major histocompatibility complex class I as a molecular marker of nontherapeutic cells and associated with decreased lung retention. Conclusions: UC-MSCs with a progenitor-like transcriptome, but not with a fibroblast-like transcriptome, provide lung protection in experimental BPD. High expression of major histocompatibility complex class I is associated with reduced therapeutic benefit. scRNA-seq may be useful to identify subsets of MSCs with superior repair capacity for clinical application.

Efficacy and Safety of Umbilical Cord-Derived Mesenchymal Stromal Cell Therapy in Preclinical Models of Sepsis: A Systematic Review and Meta-analysis.

BACKGROUND: In preclinical studies, mesenchymal stromal cells (MSCs), including umbilical cord-derived MSCs (UC-MSCs), demonstrate the ability to modulate numerous pathophysiological processes related to sepsis; however, a systematic synthesis of the literature is needed to assess the efficacy of UC-MSCs for treating sepsis. OBJECTIVE: To examine the effects of UC-MSCs on overall mortality (primary outcome) as well as on organ dysfunction, coagulopathy, endothelial permeability, pathogen clearance, and systemic inflammation (secondary outcomes) at prespecified time intervals in preclinical models of sepsis. METHODS: A systematic search was conducted on Embase, Ovid MEDLINE, and Web of Science up to June 20, 2023. Preclinical controlled studies using in vivo sepsis models with systemic UC-MSC administration were included. Meta-analyses were conducted and expressed as odds ratios (OR) and ratios of the weighted means with 95% CI for categorical and continuous data, respectively. Risk of bias was assessed with the SYRCLE tool. RESULTS: Twenty-six studies (34 experiments, n = 1258 animals) were included in this review. Overall mortality was significantly reduced with UC-MSC treatment as compared to controls (OR: 0.26, 95% CI: 0.18-0.36). At various prespecified time intervals, UC-MSCs reduced surrogate measures of organ dysfunction related to the kidney, liver, and lung; reduced coagulopathy and endothelial permeability; and enhanced pathogen clearance from multiple sites. UC-MSCs also modulated systemic inflammatory mediators. No studies were rated as low risk across all SYCLE domains. CONCLUSIONS: These results demonstrate the efficacy of UC-MSC treatment in preclinical sepsis models and highlight their potential as a therapeutic intervention for septic shock.

A preclinical systematic review and meta-analysis assessing the effect of biological sex in lipopolysaccharide-induced acute lung injury.

It is unclear what effect biological sex has on outcomes of acute lung injury (ALI). Clinical studies are confounded by their observational design. We addressed this knowledge gap with a preclinical systematic review of ALI animal studies. We searched MEDLINE and Embase for studies of intratracheal/intranasal/aerosolized lipopolysaccharide administration (the most common ALI model) that reported sex-stratified data. Screening and data extraction were conducted in duplicate. Our primary outcome was histological tissue injury and secondary outcomes included alveolar-capillary barrier alterations and inflammatory markers. We used a random-effects inverse variance meta-analysis, expressing data as standardized mean difference (SMD) with 95% confidence intervals (CIs). Risk of bias was assessed using the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool. We identified six studies involving 132 animals across 11 independent experiments. A total of 41 outcomes were extracted, with the direction of effect suggesting greater severity in males than females in 26/41 outcomes (63%). One study reported on lung histology and found that male mice exhibited greater injury than females (SMD: 1.61, 95% CI: 0.53-2.69). Meta-analysis demonstrated significantly elevated albumin levels (SMD: 2.17, 95% CI: 0.63-3.70) and total cell counts (SMD: 0.80, 95% CI: 0.27-1.33) in bronchoalveolar lavage fluid from male mice compared with female mice. Most studies had an "unclear risk of bias." Our findings suggest sex-related differences in ALI severity. However, these conclusions are drawn from a small number of animals and studies. Further research is required to address the fundamental issue of biological sex differences in LPS-induced ALI.

Safety and biodistribution of XC001 (encoberminogene rezmadenovec) gene therapy in rats: a potential therapy for cardiovascular diseases.

Adenovirus-mediated gene therapy holds promise for the treatment of cardiovascular diseases such as refractory angina. However, potential concerns around immunogenicity and vector dissemination from the target injected tissue require evaluation. This study was undertaken to evaluate the safety and biodistribution of XC001, a replication-deficient adenovirus serotype 5 vector expressing multiple isoforms of human vascular endothelial growth factor (VEGF), following direct administration into normal rat myocardium. Animals received the buffer formulation or increasing doses of XC001 (1 × 107, 2.5 × 108 or 2.5 × 109 viral particles). Based on in-life parameters (general health, body weights, clinical pathology, serum cardiac troponin I, plasma VEGF, and gross necropsy), there were no findings of clinical concern. On Day 8, intramyocardial administration of XC001 was associated with dose-related, left ventricular myocardial inflammation at injection sites, resolving by Day 30. XC001 DNA was not detected in blood at any time but was present at Day 8 around the site of injection and to a much lesser extent in the spleen, liver, and lungs, persisting at low levels in the heart and spleen until at least Day 91. These findings demonstrate that intramyocardial injection of XC001 is supported for use in human studies.

Metabolomics Analysis of Mesenchymal Stem Cell (MSC) Therapy in a Phase I Clinical Trial of Septic Shock: An Exploratory Study.

Sepsis is the result of an uncontrolled host inflammatory response to infection that may lead to septic shock with multiorgan failure and a high mortality rate. There is an urgent need to improve early diagnosis and to find markers identifying those who will develop septic shock and certainly a need to develop targeted treatments to prevent septic shock and its high mortality. Herein, we explore metabolic alterations due to mesenchymal stromal cell (MSC) treatment of septic shock. The clinical findings for this study were already reported; MSC therapy was well-tolerated and safe in patients in this phase I clinical trial. In this exploratory metabolomics study, 9 out of 30 patients received an escalating dose of MSC treatment, while 21 patients were without MSC treatment. Serum metabolomics profiling was performed to detect and characterize metabolite changes due to MSC treatment and to help determine the sample size needed for a phase II clinical trial and to define a metabolomic response to MSC treatment. Serum metabolites were measured using 1H-NMR and HILIC-MS at times 0, 24 and 72 h after MSC infusion. The results demonstrated the significant impact of MSC treatment on serum metabolic changes in a dose- and time-dependent manner compared to non-MSC-treated septic shock patients. This study suggests that plasma metabolomics can be used to assess the response to MSC therapy and that treatment-related metabolomics effects can be used to help determine the sample size needed in a phase II trial. As this study was not powered to detect outcome, how the treatment-induced metabolomic changes described in this study of MSC-treated septic shock patients are related to outcomes of septic shock in the short and long term will need to be explored in a larger adequately powered phase II clinical trial.

Biodistribution of mesenchymal stromal cell-derived extracellular vesicles administered during acute lung injury.

BACKGROUND: Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are a promising cell-free therapy for acute lung injury (ALI). To date, no studies have investigated their biodistribution in ALI or discerned the timing of administration for maximal lung targeting, which are crucial considerations for clinical translation. Our study aimed to characterize a mouse model of ALI and establish the distribution kinetics and optimal timing of MSC-EV delivery during lung injury. METHODS: MSC-EVs were isolated by ultracentrifugation alone (U/C) or tangential flow filtration with ultracentrifugation (TFF-U/C) and characterized by nanoparticle tracking analysis and western blot. A lipopolysaccharide (LPS)-induced mouse model of ALI was established to study the inflammatory response over 72 h. ALI was assessed by histological lung injury score, bronchoalveolar lavage fluid cell count and inflammatory cytokines. For biodistribution studies, ALI mice were intravenously administered fluorescently labeled MSC-EVs to determine the optimal timing of administration and organ-specific biodistribution. Live in vivo and ex vivo fluorescence imaging was conducted at various timepoints post-EV injection. RESULTS: EVs isolated by either ultracentrifugation alone or TFF-U/C displayed comparable size distribution (~ 50-350 nm) and EV marker expression (CD63/81). TFF-U/C generated a 5.4-fold higher particle concentration and 3.9-fold higher total protein when compared to ultracentrifugation alone. From the inflammatory time-course study, cell count and IL-1ß peaked in bronchoalveolar lavage fluid at 24 h after ALI induction. MSC-EVs delivered at 24 h (as opposed to 0.5 h, 5 h or 10 h) after disease induction resulted in a 2.7-4.4-fold higher lung uptake of EVs. Biodistribution studies comparing organ-specific MSC-EV uptake showed progressive lung accumulation up to 48 h post-delivery (threefold higher than the spleen/liver), with a decline at 72 h. Importantly, lung EV fluorescence at 48 h in ALI mice was significantly elevated as compared to control mice. The lung tropism of MSC-EVs was further validated as therapeutically inert EVs derived from HEK293T cells accumulated mainly to the spleen and liver with a 5.5-fold lower distribution to the lungs as compared to MSC-EVs. CONCLUSION: MSC-EVs exhibit maximal lung accumulation when administered during heightened inflammation at 24 h after ALI induction. This lung tropism suggests that MSC-EVs may serve as a practical rescue treatment for acute inflammatory respiratory conditions.

Prevention of atrial fibrillation after open-chest surgery with extracellular vesicle therapy.

Almost half of patients recovering from open-chest surgery experience atrial fibrillation (AF) that results principally from inflammation in the pericardial space surrounding the heart. Given that postoperative AF is associated with increased mortality, effective measures to prevent AF after open-chest surgery are highly desirable. In this study, we tested the concept that extracellular vesicles (EVs) isolated from human atrial explant-derived cells can prevent postoperative AF. Middle-aged female and male rats were randomized to undergo sham operation or induction of sterile pericarditis followed by trans-epicardial injection of human EVs or vehicle into the atrial tissue. Pericarditis increased the probability of inducing AF while EV treatment abrogated this effect in a sex-independent manner. EV treatment reduced infiltration of inflammatory cells and production of pro-inflammatory cytokines. Atrial fibrosis and hypertrophy seen after pericarditis were markedly attenuated by EV pretreatment, an effect attributable to suppression of fibroblast proliferation by EVs. Our study demonstrates that injection of EVs at the time of open-chest surgery shows prominent antiinflammatory effects and prevents AF due to sterile pericarditis. Translation of this finding to patients might provide an effective new strategy to prevent postoperative AF by reducing atrial inflammation and fibrosis.

Mesenchymal stem cells induce dynamic immunomodulation of airway and systemic immune cells in vivo but do not improve survival for mice with H1N1 virus-induced acute lung injury.

Introduction: Influenza A virus (IAV)-induced acute lung injury (ALI) is characterized by pronounced proinflammatory activation and respiratory lung dysfunction. In this study, we performed deep immune profiling on airway and circulating immune cells to examine the effect of immunomodulation and therapeutic outcomes of mesenchymal stem cells (MSCs) therapy in mice with IAV-induced ALI. Methods: Animals were inoculated intranasally with H1N1 IAV, followed by intravenous administration of vehicle, or human clinical-grade, bone marrow-derived MSCs 24-h later, and monitored for six days to evaluate the survival. In another set of animals, bronchoalveolar lavage (BAL) fluid and whole blood were collected three days after infection for flow or mass cytometry (CyTOF) immune profiling analysis. Results: Immune cell population and phenotypic shifts in blood were mapped by CyTOF. Increases were observed in granulocytes and myeloid-derived cells in blood from vehicle-treated animals. While MSC treatment accentuated changes in these populations, naïve B, antibody-secreting B cells, and T cells were decreased in MSC-treated animals at day 3. Compared to sham animals, IAV infection induced a significant 5.5-fold increase in BAL total cell counts, including CD4+ and CD8+ T cells, CD19+ B cells, CD11b + Ly6G + neutrophils, and CD11b + Ly6C + monocytes. MSC treatment significantly decreased BAL total cell counts in IAV-infected mice, specifically the number of infiltrating CD4+ T cells and CD11b + Ly6G + neutrophils. In contrast, there were increases in CD8+ T cells, B cells, and monocytes in the alveolar space in MSC-treated animals. Phenotypic immune cell profiling of blood and BAL revealed a significantly higher proportion of the monocyte population with the M2 phenotype (CD206) in MSC-treated animals; however, this failed to confer protective effects in the survival of infected mice or reduce viral titer in the lung. Further investigation revealed that MSCs were susceptible to IAV infection, leading to increased cell death and potentially affecting their efficacy. Conclusion: These findings provided in vivo evidence that MSCs promote the selective recruitment of immune cells to the site of infection during IAV infection, with reductions in proinflammatory phenotypes. However, MSCs offered no survival benefit in IAV-infected animals, possibly due to MSCs' H1N1 IAV susceptibility and subsequent cell death.

Single-cell transcriptomic atlas of lung microvascular regeneration after targeted endothelial cell ablation.

We sought to define the mechanism underlying lung microvascular regeneration in a model of severe acute lung injury (ALI) induced by selective lung endothelial cell ablation. Intratracheal instillation of DT in transgenic mice expressing human diphtheria toxin (DT) receptor targeted to ECs resulted in ablation of >70% of lung ECs, producing severe ALI with near complete resolution by 7 days. Using single-cell RNA sequencing, eight distinct endothelial clusters were resolved, including alveolar aerocytes (aCap) ECs expressing apelin at baseline and general capillary (gCap) ECs expressing the apelin receptor. At 3 days post-injury, a novel gCap EC population emerged characterized by de novo expression of apelin, together with the stem cell marker, protein C receptor. These stem-like cells transitioned at 5 days to proliferative endothelial progenitor-like cells, expressing apelin receptor together with the pro-proliferative transcription factor, Foxm1, and were responsible for the rapid replenishment of all depleted EC populations by 7 days post-injury. Treatment with an apelin receptor antagonist prevented ALI resolution and resulted in excessive mortality, consistent with a central role for apelin signaling in EC regeneration and microvascular repair. The lung has a remarkable capacity for microvasculature EC regeneration which is orchestrated by newly emergent apelin-expressing gCap endothelial stem-like cells that give rise to highly proliferative, apelin receptor-positive endothelial progenitors responsible for the regeneration of the lung microvasculature.

Extracellular vesicle microRNA and protein cargo profiling in three clinical-grade stem cell products reveals key functional pathways.

The cell origin-specific payloads within extracellular vesicles (EVs) mediate therapeutic bioactivity for a wide variety of stem cell types. In this study, we profiled the microRNA (miRNA) and protein cargos found within EVs produced by three clinical-grade stem cell products of different ontogenies being considered for clinical application, namely bone marrow-derived mesenchymal stromal cells (BM-MSCs), heart-derived cells (HDCs), and umbilical cord-derived MSCs (UC-MSCs). Although several miRNAs (757) and proteins (420) were found in common, each producer cell type expressed unique miRNA profiles when the most highly expressed transcripts were compared. Differential expression analysis revealed that BM-MSCs and HDCs were quite similar, while UC-MSCs had the greatest number of unique miRNAs and proteins. Despite these differences, all three EVs promoted cell adhesion/migration, immune response, platelet aggregation, protein translation/stabilization, and RNA processing. EVs from BM-MSCs were implicated in apoptosis, cell-cycle progression, collagen formation, heme pigment synthesis, and smooth muscle differentiation, while HDC and UC-MSC EVs were found to regulate complement activation, endopeptidase activity, and matrix metallopeptidases. Overall, miRNA and protein profiling reveal functional differences between three leading stem cell products. These findings provide a framework for mechanistic exploration of candidate therapeutic molecules driving the salutary effects of EVs.

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

Targeting extracellular vesicle delivery to the lungs by microgel encapsulation.

Extracellular vesicles (EVs) secreted by stem and progenitor cells have significant potential as cell-free 'cellular' therapeutics. Yet, small EVs (<200 nm) are rapidly cleared after systemic administration, mainly by the liver, presenting challenges targeting EVs to a specific organ or tissue. Microencapsulation using natural nano-porous hydrogels (microgels) has been shown to enhance engraftment and increase the survival of transplanted cells. We sought to encapsulate EVs within microgels to target their delivery to the lung by virtue of their size-based retention within the pulmonary microcirculation. Mesenchymal stromal cell (MSC) derived EVs were labelled with the lipophilic dye (DiR) and encapsulated within agarose-gelatin microgels. Endothelial cells and bone marrow derived macrophages were able to take up EVs encapsulated in microgels in vitro, but less efficiently than the uptake of free EVs. Following intrajugular administration, microgel encapsulated EVs were selectively retained within the lungs for 72h, while free EVs were rapidly cleared by the liver. Furthermore, microgel-loaded EVs demonstrated greater uptake by lung cells, in particular CD45+ immune cells, as assessed by flow cytometry compared to free EVs. Microencapsulation of EVs may be a novel tool for enhancing the targeted delivery of EVs for future therapeutic applications.

Evaluation of Lung Glucose Uptake with Fluorine-18 Fluorodeoxyglucose Positron Emission Tomography/CT in Patients with Pulmonary Arterial Hypertension and Pulmonary Hypertension Due to Left Heart Disease.

Aim: Previous studies have demonstrated increased glucose uptake by 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) in lung parenchyma in animal models or small pulmonary arterial hypertension (PAH) cohorts. However, it is not well known whether increased FDG uptake in the lung is a unique phenomenon in PAH or whether elevated pulmonary artery pressure (PAP) induces FDG uptake. Methods and results: Nineteen patients with PAH, 8 patients with pulmonary hypertension due to left heart disease (PH-LHD), and 14 age matched control subjects were included. All PH patients underwent right heart catheterization and FDG-PET. The mean standard uptake value (SUV g/mL) of FDG in each lung was obtained and average values of both lungs were calculated as mean lung FDG SUV. The correlation between hemodynamics and mean lung FDG SUV was also analyzed in PH patients. Mean PAP (mPAP) was not significantly different between PAH and PH-LHD (45±11 vs 43±5 mmHg, p=0.51). PAH patients demonstrated significantly increased mean lung FDG SUV compared with PH-LHD and controls (PAH: 0.76±0.26 vs PH-LHD: 0.51±0.12 vs controls: 0.53±0.16, p=0.0025). The mean lung FDG SUV did not correlate with mPAP either in PAH or PH-LHD. Conclusion: PAH is associated with increased lung FDG uptake indicating increased glucose utilization in the lung. This may represent metabolic shift to glycolysis and/or active inflammation in the remodeled pulmonary vasculature, and is observed to a greater extent in PAH than in patients with PH secondary to LHD and control subjects without PH.

Right Ventricular Maladaptation to Pressure Overload in Fischer Rats Is Associated With Profound Deficiency in Adenylate Kinase 1 and Impaired Ventricular Energetics.

BACKGROUND: We explored the mechanism of maladaptive right ventricular (RV) remodeling in Fischer compared with Sprague-Dawley (SD) rats exposed to pressure overload. METHODS: Pulmonary hypertension was induced by injection of the VEGFR antagonist, SU5416, followed by a 3-week exposure to hypoxia (Sugen chronic hypoxia). In vivo oxidative metabolism was assessed by RV/left ventricle ratio of [11C]acetate positron emission tomography clearance (kmono). Unbiased, global transcriptional and proteomic profiling was performed in Fischer and SD rats at baseline and after Sugen chronic hypoxia. RESULTS: All Fischer rats succumbed to RV failure by 5 weeks, whereas SD rats showed preserved RV function and 88% survival beyond 9 weeks (P<0.0001). Fischer rats exhibited increased oxidative metabolism at 4 weeks (P<0.05) and impaired RV efficiency compared with SD (work metabolic index: 52±10 versus 91±27 mmHg·mL/cm2, respectively; P<0.05), but no differences in mitochondrial complex activity. AK1 (adenylate kinase 1) was among the top 10 differentially expressed genes between Fischer and SD rats, with markedly lower RV expression in Fischer rats (FC: 3.36, P<0.05), confirmed by proteomic analysis and validated by Western blotting (>10-fold reduction, P<0.001). While whole-genome sequencing failed to reveal any coding region mutations in Fischer rats, there was a unique variant in a highly conserved upstream flanking region likely involved in the regulation of AK1 expression. CONCLUSIONS: Therefore, Fischer rats exhibit profound AK1 deficiency and inefficient cardiac energetics likely related to reduced adenosine triphosphate shuttling from the mitochondria to the contractile fibers. This represents a novel mechanism for RV failure in response to chronic increases in afterload.

Evolving Concepts in Endothelial Pathobiology of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a deadly disease, characterized by increased vascular resistance, pulmonary arteriolar loss, and occlusive arterial remodeling, leading to eventual right heart failure. Evidence increasingly points to the pulmonary endothelium as a central actor in PAH. Endothelial cell apoptosis can result directly in distal lung arteriolar pruning and indirectly in the formation of complex and occlusive arterial lesions, reflecting an imbalance between endothelial injury and repair in the development and progression of PAH. Many of the mutations implicated in PAH are in genes, which are predominantly, or solely, expressed in endothelial cells, and the endothelium is a major target for therapeutic interventions to restore BMP signaling. We explore how arterial pruning can promote the emergence of occlusive arterial remodeling mediated by ongoing endothelial injury secondary to hemodynamic perturbation and pathological increases in luminal shear stress. The emerging role of endothelial cell senescence is discussed in the transition from reversible to irreversible arterial remodeling in advanced PAH, and we review the sometimes conflicting evidence that female sex hormones can both protect or promote vascular changes in disease. Finally, we explore the contribution of the endothelium to metabolic changes and the altered inflammatory and immune state in the PAH lung, focusing on the role of excessive TGFß signaling. Given the complexity of the endothelial pathobiology of PAH, we anticipate that emerging technologies that allow the study of molecular events at a single cell level will provide answers to many of the questions raised in this review.

Poly(I:C) enhances mesenchymal stem cell control of myeloid cells from COVID-19 patients.

Mesenchymal stem cells (MSCs) are being studied for the treatment of COVID-19-associated critical illness, due to their immunomodulatory properties. Here, we hypothesized that viral mimic-priming improves MSCs' abilities to rebalance the dysregulated immune responses in COVID-19. Transcriptome analysis of poly(I:C)-primed MSCs (pIC-MSCs) showed upregulation of pathways in antiviral and immunomodulatory responses. Together with increased expression of antiviral proteins such as MX1, IFITM3, and OAS1, these changes translated to greater effector functions in regulating monocytes and granulocytes while further enhancing MSCs' ability to block SARS-CoV-2 pseudovirus entry into epithelial cells. Most importantly, the addition of pIC-MSCs to COVID-19 patient whole blood significantly reduced inflammatory neutrophils and increased M2 monocytes while enhancing their phagocytic effector function. We reveal for the first time that MSCs can be primed by Toll-like receptor 3 agonist to improve their ability to rebalance the dysregulated immune responses seen in severe SARS-CoV-2 infection.