Circulating Cell Biomarkers in Pulmonary Arterial Hypertension: Relationship with Clinical Heterogeneity and Therapeutic Response

Biomarcadores; Disfunción endotelial; Células progenitorasBiomarcadors; Disfunció endotelial; Cèl·lules progenitoresBiomarkers; Endothelial dysfunction; Progenitor cellsBackground: Endothelial dysfunction is central to PAH. In this study, we simultaneously analysed circulating levels of endothelial microvesicles (EMVs) and progenitor cells (PCs) in PAH and in controls, as biomarkers of pulmonary endothelial integrity and evaluated differences among PAH subtypes and as a response to treatment. Methods: Forty-seven controls and 144 patients with PAH (52 idiopathic, 9 heritable, 31 associated with systemic sclerosis, 15 associated with other connective tissue diseases, 20 associated with HIV and 17 associated with portal hypertension) were evaluated. Forty-four patients with scleroderma and 22 with HIV infection, but without PAH, were also studied. Circulating levels of EMVs, total (CD31+CD42b−) and activated (CD31+CD42b−CD62E+), as well as circulating PCs (CD34+CD133+CD45low) were measured by flow cytometry and the EMVs/PCs ratio was computed. In treatment-naïve patients, measurements were repeated after 3 months of PAH therapy. Results: Patients with PAH showed higher numbers of EMVs and a lower percentage of PCs, compared with healthy controls. The EMV/PC ratio was increased in PAH patients, and in patients with SSc or HIV without PAH. After starting PAH therapy, individual changes in EMVs and PCs were variable, without significant differences being observed as a group. Conclusion: PAH patients present disturbed vascular homeostasis, reflected in changes in circulating EMV and PC levels, which are not restored with PAH targeted therapy. Combined measurement of circulating EMVs and PCs could be foreseen as a potential biomarker of endothelial dysfunction in PAH.This research was funded by grants PI12/00510, PI15/00582 and PI18/00960 from the Institute of Health Carlos III (ISCiii), Spain, co-funded by the European Union (ERDF/ESF); the Catalan Society of Respiratory Medicine (SOCAP); and the Fundación Contra la Hipertensión Pulmonar (FCHP). OTC is the former recipient of a Marie Curie Post-Doctoral Fellowship Award BIOTRACK-IDIBAPS, and the current recipient of a Miguel Servet contract from ISCiii (CP17/00114)

Progenitor cell mobilisation and recruitment in pulmonary arteries in chronic obstructive pulmonary disease

BACKGROUND: Pulmonary vascular abnormalities are a characteristic feature of chronic obstructive pulmonary disease (COPD). Cigarette smoking is the most important risk factor for COPD. It is believed that its constant exposure triggers endothelial cell damage and vascular remodelling. Under pathological conditions, progenitor cells (PCs) are mobilized from the bone marrow and recruited to sites of vascular injury. The aim of the study was to investigate whether in COPD the number of circulating PCs is related to the presence of bone marrow-derived cells in pulmonary arteries and the association of these phenomena to both systemic and pulmonary endothelial dysfunction. METHODS: Thirty-nine subjects, 25 with COPD, undergoing pulmonary resection because of a localized carcinoma, were included. The number of circulating PCs was assessed by flow cytometry using a triple combination of antibodies against CD45, CD133 and CD34. Infiltrating CD45+ cells were identified by immunohistochemistry in pulmonary arteries. Endothelial function in systemic and pulmonary arteries was measured by flow-mediated dilation and adenosine diphosphate-induced vasodilation, respectively. RESULTS: COPD patients had reduced numbers of circulating PCs (p < 0.05) and increased numbers of CD45+ cells (< 0.05) in the pulmonary arterial wall than non-COPD subjects, being both findings inversely correlated (r = - 0.35, p < 0.05). In pulmonary arteries, the number of CD45+ cells correlated with the severity of vascular remodelling (r = 0.4, p = 0.01) and the endothelium-dependent vasodilation (r = - 0.3, p = 0.05). Systemic endothelial function was unrelated to the number of circulating PCs and changes in pulmonary vessels. CONCLUSION: In COPD, the decrease of circulating PCs is associated with their recruitment in pulmonary arteries, which in turn is associated with endothelial dysfunction and vessel remodelling, suggesting a mechanistic link between these phenomena. Our findings are consistent with the notion of an imbalance between endothelial damage and repair capacity in the pathogenesis of pulmonary vascular abnormalities in COPD

Remodelat vascular a les malalties pulmonars: perfil d’expressió gènica i biomarcadors

[cat] En la malaltia pulmonar obstructiva crònica (MPOC) i la hipertensió pulmonar (HP) el remodelat vascular i la disfunció endotelial són dos dels trets més característics. En el primer treball d’aquesta Tesi Doctoral s’ha descrit el perfil d’expressió gènica de les artèries pulmonars obtingudes de malalts amb MPOC segons el grau de remodelat vascular. Els resultats mostren que, tant l’expressió gènica d’angiopoietina-2 (ANGPT-2) com l’expressió proteica, es troben incrementats en aquelles artèries amb major grau de remodelat. Els resultats de l’anàlisi multivariant mostren que, junt amb l’ANGPT-2, la combinació d’altres 9 gens permeten classificar les artèries pulmonars dels malalts segons el grau de remodelat vascular amb un 96.6% d’eficàcia. D’aquests 10 gens, 5 gens mostren correlació múltiple entre ells, explicant el 52% de la variabilitat de l’engruiximent de la capa íntima. Els nivells plasmàtics d’ANGPT-2 es troben elevats en aquells malalts amb MPOC comparat amb els subjectes sans. A més a més, aquells malalts amb MPOC fumadors o amb HP associada, presenten nivells plasmàtics d’ANGPT-2 encara més incrementats. El diagnòstic, tant de la MPOC com de la HP, no avalua de manera directa l’estat endotelial dels pacients. Per aquesta raó, en aquesta Tesi Doctoral també hem avaluat biomarcadors endotelials. Específicament hem avaluat els nivells de cèl·lules progenitores (CPs) i micropartícules endotelials (EMPs). En el segon treball d’aquesta Tesi Doctoral hem descrit que els malalts amb hipertensió arterial pulmonar (HAP), però no els malalts amb hipertensió pulmonar tromboembòlica crònica (HPTEC), presenten nivells reduïts de CPs en comparació amb els controls. Donat que els malalts són reclutats en el moment del diagnòstic i, per tant, no estan tractats prèviament amb fàrmacs per la HAP, la disminució dels nivells de CPs trobats en els malats amb HAP podria ser degut a mecanismes de la pròpia malaltia. Després d’un període de 6 a 12 mesos amb teràpia específica per la HAP, els malalts amb HAP (però no els malalts amb HPTEC) mostren un augment dels nivells circulants de CPs en comparació amb els nivells basals, assolint nivells similiars als dels controls. Finalment, també hem observat que els nivells de CPs correlacionen amb la tolerància a l’esforç, és a dir, aquells malalts amb major nombre de CPs circulants són aquells que caminen més metres a la prova d’esforç. En el tercer treball d’aquesta Tesi Doctoral hem avaluat per primera vegada a la MPOC l’estat de competència vascular dels malalts mitjançant l’avaluació del dany endotelial (nivells de micropartículas endotelials (EMPs)) i la capacitat de reparació (nivells de CPs). Els resultats mostren que els malalts amb MPOC tenen un major número d’EMPs circulants que els controls, tant si es corretgeixen les dades per les variables poblacionals com no. Per la seva banda, els nivells de CPs es troben disminuïts en els malalts amb MPOC en comparació amb els subjectes fumadors. No obstant, al corretgir les dades per las variables poblacionals, es va observar que el sexe té efecte sobre els nivells de CPs. Les dones presenten menys CPs que els homes en tots els grups de l’estudi, siguent aquesta reducció significativa en el grup de malalts amb MPOC. A l’hora d’avaluar la competència vascular mitjantçant el ratio EMPs/CPs es va observar que els malalts amb MPOC presenten un ratio major comparat amb els controls. A més, aquesta incompetència vascular sembla ser deguda a la pròpia malaltia i no a l’efecte directe del consum de tabac. En dones, aquest ratio està associat a una pitjor funció pulmonar.[eng] Pulmonary vessel remodeling and endothelial dysfunction are two of the most outstanding features in chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (PH). As pulmonary vessel remodeling involves changes in smooth muscle cell proliferation, which are highly dependent on the coordinated interaction of angiogenic related growth factors, in the first study we investigated, in isolated pulmonary arteries from patients with COPD, the gene expression of 46 genes known to be modulators of the angiogenic process and/or involved in smooth muscle cell proliferation and to relate it to vascular remodeling. We conclude that an imbalance in ANGPT-2, combined with related factors such as VEGF, b-catenin, and MMP-2, may partially explain the structural derangements of the arterial wall. MMP-2 may act as a bridge channeling actions from the main fibrogenic factors. Endothelial dysfunction is key in the development of pulmonary hypertension (PH) as well as in COPD and is associated with reduced number of circulating progenitor cells. Studies to date evaluating levels of circulating progenitor cells in PH have provided conflicting results. For this reason, the aim of the second study was to assess whether levels of circulating progenitor cells in treatment-naïve patients with PAH or CTEPH differ from healthy subjects and to assess the effect of PAH-targeted therapy on the circulating levels of these progenitors. Patients with PAH, but not those with CTEPH, present reduced levels of circulating progenitor cells. PAH-targeted therapy increases levels of progenitors in PAH but not in CTEPH, suggesting different involvement of progenitor cells in the pathobiology of these pulmonary hypertensive disorders. In the third study we aimed to investigate whether COPD patients have an imbalance between EMPs to PCs compared to controls and to evaluate the effect of cigarette smoke on these circulating markers. We conclude that COPD patients present an imbalance between endothelial damage and repair capacity that might explain the frequent concurrence of cardiovascular disorders. Factors related to the disease itself and gender, rather than cigarette smoking, may account for this imbalance

Effects of exercise training on circulating biomarkers of endothelial function in pulmonary arterial hypertension

Introduction: In stable patients with pulmonary arterial hypertension (PAH), pulmonary rehabilitation (PR) is an effective, safe and cost-effective non-pharmacological treatment. However, the effects of PR on vascular function have been poorly explored. This study aimed to compare the amounts of circulating progenitor cells (PCs) and endothelial microvesicles (EMVs) in patients with PAH before and after 8 weeks of endurance exercise training as markers of vascular competence. Methods: A prospective study of 10 consecutive patients with PAH that successfully finished a PR program (8 weeks) was carried out before and after this intervention. Levels of circulating PCs defined as CD34+CD45low progenitor cells and levels of EMVs (CD31+ CD42b-) were measured by flow cytometry. The ratio of PCs to EMVs was taken as a measure of the balance between endothelial damage and repair capacity. Results: All patients showed training-induced increases in endurance time (mean change 287 s). After PR, the number of PCs (CD34+CD45low/total lymphocytes) was increased (p < 0.05). In contrast, after training, the level of EMVs (CD31+ CD42b-/total EMVs) was reduced. The ratio of PCs to EMVs was significantly higher after training (p < 0.05). Conclusion: Our study shows, for the first time, that endurance exercise training in patients with stable PAH has a positive effect, promoting potential mechanisms of damage/repair in favor of repair. This effect could contribute to a positive hemodynamic and clinical response