Prognostic Significance of Proteomics-Discovered Circulating Inflammatory Biomarkers in Patients With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) ultimately leads to right ventricular failure and premature death. The identification of circulating biomarkers with prognostic utility is considered a priority. As chronic inflammation is recognized as key pathogenic driver, we sought to identify inflammation-related circulating proteins that add incremental value to current risk stratification models for long-term survival in patients with PAH. METHODS AND RESULTS: Plasma levels of 384 inflammatory proteins were measured with the proximity extension assay technology in patients with PAH (n=60) and controls with normal hemodynamics (n=28). Among these, 51 analytes were significantly overexpressed in the plasma of patients with PAH compared with controls. Cox proportional hazard analyses and C-statistics were performed to assess the prognostic value and the incremental prognostic value of differentially expressed proteins. A panel of 6 proteins (CRIM1 [cysteine rich transmembrane bone morphogenetic protein regulator 1], HGF [hepatocyte growth factor], FSTL3 [follistatin-like 3], PLAUR [plasminogen activator, urokinase receptor], CLSTN2 [calsyntenin 2], SPON1 [spondin 1]) were independently associated with death/lung transplantation at the time of PAH diagnosis after adjustment for the 2015 European Society of Cardiology/European Respiratory Society guidelines, the REVEAL (Registry to Evaluate Early and Long-Term PAH Disease Management) 2.0 risk scores, and the refined 4-strata risk assessment. CRIM1, PLAUR, FSTL3, and SPON1 showed incremental prognostic value on top of the predictive models. As determined by Western blot, FSTL3 and SPON1 were significantly upregulated in the right ventricle of patients with PAH and animal models (monocrotaline-injected and pulmonary artery banding-subjected rats). CONCLUSIONS: In addition to revealing new actors likely involved in cardiopulmonary remodeling in PAH, our screening identified promising circulating biomarkers to improve risk prediction in PAH, which should be externally confirmed.

Exploring Integrin α5ß1 as a Potential Therapeutic Target for Pulmonary Arterial Hypertension: Insights from Comprehensive Multicenter Preclinical Studies.

Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PA) and progressive increase in pulmonary vascular resistance (PVR) leading to right ventricular (RV) failure. Although several drugs are approved for the treatment of PAH, mortality remains high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. We found that the arginine-glycine-aspartate (RGD)-binding integrin α5ß1 is upregulated in PA endothelial cells (PAEC) and PA smooth muscle cells (PASMC) from PAH patients and remodeled PAs from animal models. Blockade of the integrin α5ß1 or depletion of the α5 subunit resulted in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. Using a novel small molecule integrin inhibitor and neutralizing antibodies, we demonstrated that α5ß1 integrin blockade attenuates pulmonary vascular remodeling and improves hemodynamics and RV function in multiple preclinical models. Our results provide converging evidence to consider α5ß1 integrin inhibition as a promising therapy for pulmonary hypertension. One sentence summary: The α5ß1 integrin plays a crucial role in pulmonary vascular remodeling.

RV-specific Targeting of Snai1 Rescues Pulmonary Hypertension-induced Right Ventricular Failure by Inhibiting EndMT and Fibrosis via LOXL2 Mediated Mechanism.

Background: Pulmonary hypertension (PH)-induced right ventricular (RV) failure (PH-RVF) is a significant prognostic determinant of mortality and is characterized by RV hypertrophy, endothelial-to-mesenchymal transition (EndMT), fibroblast-to-myofibroblast transition (FMT), fibrosis, and extracellular matrix (ECM)-remodeling. Despite the importance of RV function in PH, the mechanistic details of PH-RVF, especially the regulatory control of RV EndMT, FMT, and fibrosis, remain unclear. The action of transcription factor Snai1 is shown to be mediated through LOXL2 recruitment, and their co-translocation to the nucleus, during EndMT progression. We hypothesize that RV EndMT and fibrosis in PH-RVF are governed by the TGFß1-Snai1-LOXL2 axis. Furthermore, targeting Snai1 could serve as a novel therapeutic strategy for PH-RVF. Methods: Adult male Sprague Dawley rats (250-300g) received either a single subcutaneous injection of Monocrotaline (MCT, 60mg/kg, n=9; followed for 30-days) or Sugen (SU5416 20mg/kg, n=9; 10% O 2 hypoxia for 3-weeks followed by normoxia for 2-weeks) or PBS (CTRL, n=9). We performed secondary bioinformatics analysis on the RV bulk RNA-Seq data from MCT, SuHx, and PAB rats and human PH-PVF. We validated EndMT and FMT and their association with Snai1 and LOXL2 in the RVs of MCT and SuHx rat models and human PH-RVF using immunofluorescence, qPCR, and Western blots. For in vivo Snai1 knockdown (Snai1-KD), MCT-rats either received Snai1-siRNA (n=7; 5nM/injection every 3-4 days; 4-injections) or scramble (SCRM-KD; n=7) through tail vein from day 14-30 after MCT. Echocardiography and catheterization were performed terminally. Bulk RNASeq and differential expression analysis were performed on Snai1- and SCRM-KD rat RVs. In vitro Snai1-KD was performed on human coronary artery endothelial cells (HCAECs) and human cardiac fibroblasts (HCFs) under hypoxia+TGFß1 for 72-hrs. Results: PH-RVF had increased RVSP and Fulton index and decreased RV fractional area change (RVFAC %). RV RNASeq demonstrated EndMT as the common top-upregulated pathway between rat (MCT, SuHx, and PAB) and human PH-RVF. Immunofluorescence using EndMT- and FMT-specific markers demonstrated increased EndMT and FMT in RV of MCT and SuHx rats and PH-RVF patients. Further, RV expression of TGFß1, Snai1, and LOXL2 was increased in MCT and SuHx. Nuclear co-localization and increased immunoreactivity, transcript, and protein levels of Snai1 and LOXL2 were observed in MCT and SuHx rats and human RVs. MCT rats treated with Snai1-siRNA demonstrated decreased Snai1 expression, RVSP, Fulton index, and increased RVFAC. Snai1-KD resulted in decreased RV-EndMT, FMT, and fibrosis via a LOXL2-dependent manner. Further, Snai1-KD inhibited hypoxia+TGFß1-induced EndMT in HCAECs and FMT in HCFs in vitro by decreasing perinuclear/nuclear Snai1+LOXL2 expression and co-localization. Conclusions: RV-specific targeting of Snai1 rescues PH-RVF by inhibiting EndMT and Fibrosis via a LOXL2-mediated mechanism.

Managing Pulmonary Arterial Hypertension With Cardiopulmonary Comorbidities.

Pulmonary arterial hypertension (PAH) and pulmonary hypertension associated with left-sided heart and lung diseases are most commonly easily discriminated and treated accordingly. With the changing epidemiology of PAH, however, a growing proportion of patients at the time of diagnosis present with comorbidities of varying severity. In addition to classical PAH, two distinct phenotypes have emerged: a heart failure with preserved ejection fraction-like phenotype and a lung phenotype. Importantly, the evidence supporting the currently proposed treatment algorithm for PAH has been generated mainly from PAH trials in which patients with cardiopulmonary comorbidities have been underrepresented or excluded. As a consequence, the best therapeutic approach for patients with common PAH with cardiopulmonary comorbidities remains largely unknown and requires further investigation. The present article reviews the relevant literature on the topic and describes the authors' views on the current therapeutic approach for these patients.

Deaths induced by compassionate use of hydroxychloroquine during the first COVID-19 wave: an estimate.

BACKGROUND: During the first wave of COVID-19, hydroxychloroquine (HCQ) was used off-label despite the absence of evidence documenting its clinical benefits. Since then, a meta-analysis of randomised trials showed that HCQ use was associated with an 11% increase in the mortality rate. We aimed to estimate the number of HCQ-related deaths worldwide. METHODS AND FINDINGS: We estimated the worldwide in-hospital mortality attributable to HCQ use by combining the mortality rate, HCQ exposure, number of hospitalised patients, and the increased relative risk of death with HCQ. The mortality rate in hospitalised patients for each country was calculated using pooled prevalence estimated by a meta-analysis of published cohorts. The HCQ exposure was estimated using median and extreme estimates from the same systematic review. The number of hospitalised patients during the first wave was extracted from dedicated databases. The systematic review included 44 cohort studies (Belgium: k = 1, France: k = 2, Italy: k = 12, Spain: k = 6, Turkey: k = 3, USA: k = 20). HCQ prescription rates varied greatly from one country to another (range 16-84%). Overall, using median estimates of HCQ use in each country, we estimated that 16,990 HCQ-related in-hospital deaths (range 6267-19256) occurred in the countries with available data. The median number of HCQ-related deaths in Belgium, Turkey, France, Italy, Spain, and the USA was 240 (range not estimable), 95 (range 92-128), 199 (range not estimable), 1822 (range 1170-2063), 1895 (range 1475-2094) and 12739 (3244- 15570), respectively. CONCLUSIONS: Although our estimates are limited by their imprecision, these findings illustrate the hazard of drug repurposing with low-level evidence.

Hypusine Signaling Promotes Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.

Rationale: The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a posttranslational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) caused by excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. Objectives: To characterize the role of hypusine signaling in PAH. Methods: Molecular, genetic, and pharmacological approaches were used both in vitro and in vivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. Measurements and Main Results: Hypusine forming enzymes-deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH)-and hypusinated eukaryotic translation factor 5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using N1-guanyl-1,7-diaminoheptane or shRNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice, and its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. With mass spectrometry, hypusine signaling is shown to promote the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. Conclusions: These findings support inhibiting hypusine signaling as a potential treatment for PAH.

Right Ventricle and Epigenetics: A Systematic Review.

There is an increasing recognition of the crucial role of the right ventricle (RV) in determining the functional status and prognosis in multiple conditions. In the past decade, the epigenetic regulation (DNA methylation, histone modification, and non-coding RNAs) of gene expression has been raised as a critical determinant of RV development, RV physiological function, and RV pathological dysfunction. We thus aimed to perform an up-to-date review of the literature, gathering knowledge on the epigenetic modifications associated with RV function/dysfunction. Therefore, we conducted a systematic review of studies assessing the contribution of epigenetic modifications to RV development and/or the progression of RV dysfunction regardless of the causal pathology. English literature published on PubMed, between the inception of the study and 1 January 2023, was evaluated. Two authors independently evaluated whether studies met eligibility criteria before study results were extracted. Amongst the 817 studies screened, 109 studies were included in this review, including 69 that used human samples (e.g., RV myocardium, blood). While 37 proposed an epigenetic-based therapeutic intervention to improve RV function, none involved a clinical trial and 70 are descriptive. Surprisingly, we observed a substantial discrepancy between studies investigating the expression (up or down) and/or the contribution of the same epigenetic modifications on RV function or development. This exhaustive review of the literature summarizes the relevant epigenetic studies focusing on RV in human or preclinical setting.

Automated O2 Titration Alone or With High-Flow Nasal Cannula During Walking Exercise in Chronic Lung Diseases.

BACKGROUND: Exercise-induced O2 desaturation contributes to dyspnea and exercise intolerance in various respiratory diseases. This study assessed whether automated O2 titration was superior to fixed-flow O2 to improve exertional dyspnea and walking exercise endurance. We also aimed at evaluating possible additive effects of high-flow nasal cannula coupled with automated O2 titration on these outcomes. METHODS: Subjects with chronic respiratory diseases and exercise-induced desaturation performed a 3-min constant-speed shuttle test (CSST) and an endurance shuttle walking test (ESWT) with either (1) fixed-flow O2, (2) automated O2 titration targeting an SpO2 of 94% (± 2%), and (3) automated O2 titration + high-flow nasal cannula according to a randomized sequence. The main outcome was Borg dyspnea score at the end of the 3-min CSST. Secondary outcomes included endurance time and dyspnea during ESWT and oxygenation status during exercise. RESULTS: Ten subjects with COPD, 10 with interstitial lung disease, 5 with pulmonary hypertension, and 3 with cystic fibrosis completed the study. Compared to fixed-flow O2, automated O2 titration did not reduce dyspnea at the end of the 3-min CSST. Endurance time during the ESWT was prolonged with automated O2 titration (mean difference 298 [95% CI 205-391] s, P < .001), and dyspnea at isotime was reduced. No further improvement was noted when high-flow nasal cannula was added to automated O2 titration. Compared to fixed-flow O2, O2 flows were higher with automated O2 titration, resulting in better oxygenation. CONCLUSIONS: Automated O2 titration was superior to fixed-flow O2 to alleviate dyspnea and improve exercise endurance during the ESWT in subjects with a variety of chronic respiratory diseases. Adding high-flow nasal cannula to automated O2 titration provided no further benefits.

Effect of reverse Trendelenburg position and positive pressure ventilation on safe non-hypoxic apnea period in obese, a randomized-control trial.

PURPOSE: There is an elevated incidence of hypoxemia during the airway management of the morbidly obese. We aimed to assess whether optimizing body position and ventilation during pre-oxygenation allow a longer safe non-hypoxic apnea period (SNHAP). METHODS: Fifty morbidly obese patients were recruited and randomized for this study. Patients were positioned and preoxygenated for three minutes in the ramp position associated with spontaneous breathing without additional CPAP or PEEP (RP/ZEEP group) or in the reverse Trendelenburg position associated with pressure support ventilation mode with pressure support of 8 cmH2O and an additional 10 cmH2O of PEEP while breathing spontaneously (RT/PPV group) according to randomization. RESULTS: The SNHAP was significantly longer in the RT/PPV group (258.2 (55.1) vs. 216.7 (42.3) seconds, p = 0.005). The RT/PPV group was also associated to a shorter time to obtain a fractional end-tidal oxygen concentration (FEtO2) of 0.90 (85.1(47.8) vs 145.3(40.8) seconds, p < 0.0001), a higher proportion of patients that reached the satisfactory FEtO2 of 0.90 (21/24, 88% vs. 13/24, 54%, p = 0.024), a higher FEtO2 during preoxygenation (0.91(0.05) vs. 0.89(0.01), p = 0.003) and a faster return to 97% oxygen saturation after ventilation resumption (69.8 (24.2) vs. 91.4 (39.2) seconds, p = 0.038). CONCLUSION: In the morbidly obese population, RT/PPV, compared to RP/ZEEP, lengthens the SNHAP, decreases the time to obtain optimal preoxygenation conditions, and allows a faster resuming of secure oxygen saturation. The former combination allows a more significant margin of time for endotracheal intubation and minimizes the risk of hypoxemia in this highly vulnerable population. TRIAL REGISTRATION: NCT02590406, 29/10/2015.

The future of group 2 pulmonary hypertension: Exploring clinical trials and therapeutic targets.

Pulmonary hypertension due to left heart disease (PH-LHD) or group 2 PH is the most common and lethal form of PH, occurring secondary to left ventricular systolic or diastolic heart failure (HF), left-sided valvular diseases, and congenital abnormalities. It is subdivided into isolated postcapillary PH (IpcPH) and combined pre- and post-capillary PH (CpcPH), with the latter sharing many similarities with group 1 PH. CpcPH is associated with worse outcomes and increased morbidity and mortality when compared to IpcPH. Although IpcPH can be improved by treatment of the underlying LHD, CpcPH is an incurable disease for which no specific treatment exists, likely due to the lack of understanding of its underlying mechanisms. Furthermore, drugs approved for PAH are not recommended for group 2 PH, as they are either ineffective or even deleterious. With this major unmet medical need, a better understanding of mechanisms and the identification of effective treatment strategies for this deadly condition are urgently needed. This review presents relevant background of the molecular mechanisms underlying PH-LHD that could translate into innovative therapeutic targets and explores novel targets currently being evaluated in clinical trials.

Patients' perceptions on clinical trials outcomes in pulmonary arterial hypertension therapy.

The value placed by patients and their caregivers on the components of composite outcomes in pulmonary arterial hypertension (PAH) remains unknown. We surveyed the importance of these outcomes from a patients' and caregivers' perspective, with participants (n=335, including 257 patients with PAH) rating individual components defining clinical worsening in PAH trials as of critical, major, mild-to-moderate or minor importance. Most outcomes were considered of major or mild-to-moderate importance to patients. Death was the only outcome considered of critical importance. Perceptions of clinical outcomes varied between patients and caregivers. Integrating patients' perception in the elaboration of clinical trials is essential.

Targeting Wnt-ß-Catenin-FOSL Signaling Ameliorates Right Ventricular Remodeling.

BACKGROUND: The ability of the right ventricle (RV) to adapt to an increased pressure afterload determines survival in patients with pulmonary arterial hypertension. At present, there are no specific treatments available to prevent RV failure, except for heart/lung transplantation. The wingless/int-1 (Wnt) signaling pathway plays an important role in the development of the RV and may also be implicated in adult cardiac remodeling. METHODS: Molecular, biochemical, and pharmacological approaches were used both in vitro and in vivo to investigate the role of Wnt signaling in RV remodeling. RESULTS: Wnt/ß-catenin signaling molecules are upregulated in RV of patients with pulmonary arterial hypertension and animal models of RV overload (pulmonary artery banding-induced and monocrotaline rat models). Activation of Wnt/ß-catenin signaling leads to RV remodeling via transcriptional activation of FOSL1 and FOSL2 (FOS proto-oncogene [FOS] like 1/2, AP-1 [activator protein 1] transcription factor subunit). Immunohistochemical analysis of pulmonary artery banding -exposed BAT-Gal (ß-catenin-activated transgene driving expression of nuclear ß-galactosidase) reporter mice RVs exhibited an increase in ß-catenin expression compared with their respective controls. Genetic inhibition of ß-catenin, FOSL1/2, or WNT3A stimulation of RV fibroblasts significantly reduced collagen synthesis and other remodeling genes. Importantly, pharmacological inhibition of Wnt signaling using inhibitor of PORCN (porcupine O-acyltransferase), LGKK-974 attenuated fibrosis and cardiac hypertrophy leading to improvement in RV function in both, pulmonary artery banding - and monocrotaline-induced RV overload. CONCLUSIONS: Wnt- ß-Catenin-FOSL signaling is centrally involved in the hypertrophic RV response to increased afterload, offering novel targets for therapeutic interference with RV failure in pulmonary hypertension.

G9a/GLP Targeting Ameliorates Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling of small pulmonary arteries (PAs) causing sustained elevation of PA pressure, right ventricular failure, and death. Similar to cancer cells, PA smooth muscle cells (PASMCs), which play a key role in pulmonary vascular remodeling, have adopted multiple mechanisms to sustain their survival and proliferation in the presence of stress. The histone methyltransferase G9a and its partner protein GLP (G9a-like protein) have been shown to exert oncogenic effects and to serve as a buffer against an exaggerated transcriptional response. Therefore, we hypothesized that upregulation of G9a and GLP in PAH plays a pivotal role in pulmonary vascular remodeling by maintaining the abnormal phenotype of PAH-PASMCs. We found that G9a is increased in PASMCs from patients with PAH as well as in remodeled PAs from animal models. Pharmacological inhibition of G9a/GLP activity using BIX01294 and UNC0642 significantly reduced the prosurvival and proproliferative potentials of cultured PAH-PASMCs. Using RNA sequencing, further exploration revealed that G9a/GLP promotes extracellular matrix production and affords protection against the negative effects of an overactive stress response. Finally, we found that therapeutic treatment with BIX01294 reduced pulmonary vascular remodeling and lowered mean PA pressure in fawn-hooded rats. Treatment of Sugen/hypoxia-challenged mice with BIX01294 also improved pulmonary hemodynamics and right ventricular function. In conclusion, these findings indicate that G9a/GLP inhibition may represent a new therapeutic approach in PAH.

Transbronchial Lung Cryobiopsy and Surgical Lung Biopsy: A Prospective Multi-Centre Agreement Clinical Trial (CAN-ICE).

Rationale: Transbronchial cryobiopsy (TBCB) for the diagnosis of interstitial lung disease (ILD) has shown promising results, but prospective studies with matched surgical lung biopsy (SLB) have yielded conflicting results. Objectives: We aimed to assess within- and between-center diagnostic agreement between TBCB and SLB at both the histopathologic and multidisciplinary discussion (MDD) levels in patients with diffuse ILD. Methods: In a multicenter prospective study, we performed matched TBCB and SLB in patients referred for SLB. After a blinded review by three pulmonary pathologists, all cases were reviewed by three independent ILD teams in an MDD. MDD was performed first with TBCB, then with SLB in a second session. Within-center and between-center diagnostic agreement was evaluated using percentages and correlation coefficients. Measurements and Main Results: Twenty patients were recruited and underwent contemporaneous TBCB and SLB. Within-center diagnostic agreement between TBCB-MDD and SLB-MDD was reached in 37 of the 60 (61.7%) paired observations, resulting in a Cohen's κ value of 0.46 (95% confidence interval [CI], 0.29-0.63). Diagnostic agreement increased among high-confidence or definitive diagnoses on TBCB-MDD (21 of 29 [72.4%]), but not significantly, and was more likely among cases with SLB-MDD diagnoses of idiopathic pulmonary fibrosis than fibrotic hypersensitivity pneumonitis (13 of 16 [81.2%] vs. 16 of 31 [51.6%]; P = 0.047). Between-center agreement for cases was markedly higher for SLB-MDD (κ = 0.71 [95% CI, 0.52-0.89]) than TBCB-MDD (κ = 0.29 [95% CI, 0.09-0.49]). Conclusions: This study demonstrated moderate TBCB-MDD and SLB-MDD diagnostic agreement for ILD, while between-center agreement was fair for TBCB-MDD and substantial for SLB-MDD. Clinical trial registered with www.clinicaltrials.gov (NCT02235779).

Priorities for pulmonary hypertension research: A James Lind Alliance priority setting partnership.

Pulmonary hypertension (PH) is a rare condition associated with significant morbidity and mortality. The priorities for future research in PH according to patients, caregivers, and clinicians have not been established. We performed a James Lind Alliance priority setting partnership in Canada. An initial survey of 249 respondents (76% patients and/or caregivers, 12.5% clinicians) generated 1588 questions, which were combined into 187 summary questions. An evidence check identified 352 systematic reviews and guidelines which were mapped to the summary questions, which determined that 157 summary questions were unanswered by existing research. An interim prioritisation survey (240 respondents, 66% patients and/or caregivers, 18% clinicians) asked respondents to choose which of the 157 summary questions were among the 30-40 most important. In a final workshop patients, caregivers, and clinicians discussed and ranked the top 25 questions from the interim survey to identify the Top 10 PH research priorities. These results will inform researchers and funding bodies about patient, caregiver, and clinician priorities for future research on PH.