Stefania Ghilardi

Surfactant-Derived Proteins as Markers of Alveolar Membrane Damage in Heart Failure

Background In heart failure (HF) alveolar-capillary membrane is abnormal. Surfactant-derived proteins (SPs) and plasma receptor for advanced-glycation-end-products (RAGE) have been proposed as lung damage markers. Methods Eighty-nine chronic HF and 17 healthy subjects were evaluated by echocardiography, blood parameters, carbon monoxide lung diffusion (DLCO) and cardiopulmonary exercise test. We measured immature SP-B, mature SP-B, SP-A, SP-D and RAGE plasma levels. Results Immature SP-B (arbitrary units), mature SP-A (ng/ml) and SP-D (ng/ml), but not mature SP-B (ng/ml) and RAGE (pg/ml) levels, were higher in HF than in controls [immature SP-B: 15.6 (13.1, 75th–25th interquartile range) Vs. 11.1 (6.4), p<0.01; SP-A, 29.6 (20.1) Vs. 18.3 (13.5), p = 0.01; SP-D: 125 (90) Vs. 78 (58), p<0.01]. Immature SP-B, SP-A, SP-D and RAGE values were related to DLCO, peak oxygen consumption, ventilatory efficiency, and brain natriuretic peptide (BNP), whereas plasma mature SP-B was not. The DLCO Vs. immature SP-B correlation was the strongest one. At multivariate analysis, RAGE was associated to age and creatinine, SP-A to DLCO and BNP, SP-D to BNP, mature SP-B to DLCO and creatinine, and immature SP-B only but strongly to DLCO. Conclusions Immature SP-B is the most reliable biological marker of alveolar-capillary membrane function in HF.

Atorvastatin reduces long pentraxin 3 expression in vascular cells by inhibiting protein geranylgeranylation.

BACKGROUND The long pentraxin PTX3 is an acute-phase multi-functional protein that might play both positive and detrimental effects under different pathophysiological conditions. We previously showed that statins down-regulate the release of PTX3 in human endothelial cells (ECs). The present study investigated the mechanism mediating this effect, its occurrence in other cells involved in atherogenesis, and whether it takes place in experimental atherosclerosis. METHODS AND RESULTS We found that atorvastatin (1-5 μmol/L) decreased the production and release of PTX3 in human ECs through a post-transcriptional effect. Co-incubation with mevalonate or geranylgeranyl pyrophosphate prevented this effect. Direct blockade of geranylgeranyl transferase I by GGTI-286, treatment with the Rac inhibitor NSC23766 or silencing of the geranylgeranylated GTPase Rac2 by siRNA closely mimicked the action of atorvastatin. In contrast, inactivation of other geranylgeranylated proteins such as RhoA, RhoB, and RhoC or Rac1 did not affect PTX3 release. In addition, we found that atorvastatin also decreased PTX3 secretion in aortic SMCs through a mechanism likely dependent on protein geranylgeranylation, while no effect was observed in monocytes. Finally, we found that atherosclerotic lesions from cholesterol-fed rabbits treated with atorvastatin (2.5 mg/kg/day for 8 weeks) showed less immunoreactive PTX3 than lesions from control animals. CONCLUSIONS Results suggest that statins may interfere with PTX3 expression in vascular cells via inhibition of protein geranylgeranylation. Since PTX3 is increasingly regarded as an important mediator of the inflammatory response underlying atherosclerosis and its complications, these results highlight the need for further studies of the role of PTX3 and its potential pharmacological modulation in cardiovascular disease.

Plasma immature form of surfactant protein type B correlates with prognosis in patients with chronic heart failure. A pilot single-center prospective study

BACKGROUND Gas exchange abnormalities are part of the heart failure (HF) syndrome and growing interest raised on possible biomarkers of alveolar-capillary unit damage. The present pilot single-center study sought to investigate the prognostic values of circulating surfactant protein type B (SP-B) in a cohort of systolic HF patients. METHODS One hundred and fifty-one HF stable outpatients and 37 healthy subjects underwent a full clinical assessment, including pulmonary function and lung diffusion for carbon monoxide (DLco), maximal cardiopulmonary exercise test and measurements for both circulating immature and mature forms of SP-B. Study end-points were hospitalization due to HF worsening and cardiovascular mortality. RESULTS Immature SP-B, but not the mature form, was significantly higher in HF patients than in controls and was independently related to DLco, peak oxygen uptake and ventilatory efficiency. During the follow-up (median: 995 days; interquartile range: 739-1247 days), 97 patients experimented at least one HF hospitalization and 9 died for cardiovascular causes. At univariate analysis immature SP-B levels were significantly related to both cardiovascular death (p=0.033) and HF hospitalization (p<0.001). At multivariate analysis, immature SP-B levels remained independently associated to HF hospitalization (hazard ratio: 2.304; 95% confidence interval 1.858-3.019; p<0.001). CONCLUSIONS Present data confirm a strong relationship between circulating immature SP-B levels, gas exchange abnormalities and exercise limitations in stable HF as well as they are consistent with the use of immature SP-B in HF clinical risk assessment. Larger prospective studies are needed to confirm its prognostic role as well as to evaluate whether immature SP-B plasma concentration varies in response to specific treatment.

D-dimer is associated with arterial and venous coronary artery bypass graft occlusion

Objective In this observational prospective study, we assessed the role of clinical variables and circulating biomarkers in graft occlusion at 18 months to identify a signature for graft occlusion. Methods A total of 330 patients undergoing primary elective coronary artery bypass grafting were enrolled. Blood collection for biomarker assessment was performed before surgery and discharge. Patients were then scheduled to undergo coronary computed tomography angiography at 18 months follow‐up, and 179 patients underwent coronary computed tomography angiography 18 ± 2 months postoperatively. Results There were 46 of 503 (9.1%) occluded grafts; of these, 29 (63%) were venous and 17 (37%) were arterial grafts; overall, 43 of 179 patients (24%) had at least 1 occluded graft. Logistic mixed effects model assessing independent factors associated with graft occlusion identified that lower D‐dimer levels at baseline (odds ratio [OR], 2.58; 95% confidence interval [CI], 1.36‐4.89; P = .00) and total protein content at discharge (OR, 1.09; 95% CI, 1.01‐1.19; P = .028) were related to overall graft occlusion at follow‐up, along with an arterial graft other than the left internal thoracic artery (OR, 2.92; 95% CI, 1.24‐6.9; P = .078); moreover, a venous graft emerged was possibly associated with graft occlusion (OR, 1.51; 95% CI, 0.95‐2.39; P = .078). By separately analyzing saphenous vein and arterial grafts, D‐dimer levels (OR, 2.67; 95% CI, 1.15‐6.2; P = .022 and OR, 2.5; 95% CI, 1.01‐7.0; P = .05 for venous and arterial graft, respectively) were still associated with arterial and venous graft occlusion at follow‐up. Conclusions We identified D‐dimer as a biomarker associated with arterial and venous grafts occlusion. This may help stratify patients at risk of graft failure and identify new molecular targets to prevent this complication.

Proteomics of tissue factor silencing in cardiomyocytic cells reveals a new role for this coagulation factor in splicing machinery control.

UNLABELLED It has long been known that Tissue Factor (TF) plays a role in blood coagulation and has a direct thrombotic action that is closely related to cardiovascular risk, but it is becoming increasingly clear that it has a much wider range of biological functions that range from inflammation to immunity. It is also involved in maintaining heart haemostasis and structure, and the observation that it is down-regulated in the myocardium of patients with dilated cardiomyopathy suggests that it influences cell-to-cell contact stability and contractility, and thus contributes to cardiac dysfunction. However, the molecular mechanisms underlying these coagulation-independent functions have not yet been fully elucidated. In order to analyse the influence of TF on the cardiomyocitic proteome, we used functional biochemical approaches incorporating label-free quantitative proteomics and gene silencing, and found that this provided a powerful means of identifying a new role for TF in regulating splicing machinery together with the expression of several proteins of the spliceosome, and mRNA metabolism with a considerable impact on cell viability. BIOLOGICAL SIGNIFICANCE In this study, using quantitative proteomics and functional biochemical approaches, we define for the first time that, in addition to its primary role in blood coagulation, Tissue Factor also plays a novel role in regulating cell splicing machinery, with a relevant impact on cell survival. This new function may help to explain the wide range of biological activities of TF, and thus provide fruitful clues for developing new strategies for treating human diseases in which TF is dysregulated.

Data for proteomic analysis of murine cardiomyocytic HL-1 cells treated with siRNA against tissue factor.

This data article is related to the research article entitled Proteomics of Tissue Factor silencing in cardiomyocytic cells reveals a new role for this coagulation factor in splicing machinery control by Lento et al. [1]. Tissue Factor (TF) is a key player in the coagulation cascade, but it has additional functions ranging from angiogenesis, tumour invasion and, in the heart, the maintenance of the integrity of cardiac cells. This article reports the nano-LC–MSE analysis of the cardiomyocytic HL-1 cell line proteome and describes the results obtained from a Gene Ontology analysis of those proteins affected by TF-gene silencing.

Surfactant proteins changes after acute hemodynamic improvement in patients with advanced chronic heart failure treated with Levosimendan

Alveolar-capillary membrane evaluated by carbon monoxide diffusion (DLCO) plays an important role in heart failure (HF). Surfactant Proteins (SPs) have also been suggested as a worthwhile marker. In HF, Levosimendan improves pulmonary hemodynamics and reduces lung fluids but associated SPs and DLCO changes are unknown. Sixty-five advanced HF patients underwent spirometry, cardiopulmonary exercise test (CPET) and SPs determination before and after Levosimendan. Levosimendan caused natriuretic peptide-B (BNP) reduction, peakVO2 increase and VE/VCO2 slope reduction. Spirometry improved but DLCO did not. SP-A, SP-D and immature SP-B reduced (73.7 ± 25.3 vs. 66.3 ± 22.7 ng/mL*, 247 ± 121 vs. 223 ± 110 ng/mL*, 39.4 ± 18.7 vs. 34.4 ± 17.9AU*, respectively); while mature SP-B increased (424 ± 218 vs. 461 ± 243 ng/mL, * = p < 0.001). Spirometry, BNP and CPET changes suggest hemodynamic improvement and lung fluid reduction. SP-A, SP-D and immature SP-B reduction indicates a reduction of inflammatory stress; conversely mature SP-B increase suggests alveolar cell function restoration. In conclusion, acute lung fluid reduction is associated with SPs but not DLCO changes. SPs are fast responders to alveolar-capillary membrane condition changes.

Diving and pulmonary physiology: Surfactant binding protein, lung fluid and cardiopulmonary test changes in professional divers

Alteration of breathing pattern ranging from an increase of respiratory rate to overt hyperventilation during and after SCUBA diving is frequently reported and is associated with intrathoracic fluid overload. This study was undertaken to assess breathing efficiency after diving and the association with damage of alveolar cells. Ventilation efficiency (VE/VCO2) during maximal cardiopulmonary exercise test (CPET) before and 2h after a standard protocol dive has been analyzed in twelve professional males divers (39.5±10.5years). Furthermore, within 30min from surfacing, subjects underwent blood sample for surfactant derived proteins (SPs) determination, while thoracic ultrasound was performed at 30, 60, 90 and 120min. Dive consisted in a single quick descend to 18m of sea water, a 47min bottom stay and a direct ascent to the surface. CPET showed a preserved exercise performance with an increase of VE/VCO2 after diving (21.4±2.9 vs. 22.9±3.3, p<0.05). Mature SP-B increased while other SPs were unchanged. Ultrasound lung comets (ULC) were high in the first post-dive evaluation with a significant, but not complete, progressive reduction at 120min after surfacing. In conclusion we showed that, after a single dive, lung fluid increased with an increase of ventilation inefficiency and of the mature form of SP-B.

Optimized protocol for the extraction of proteins from the human mitral valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

Normal human mitral valve proteome: A preliminary investigation by gel‐based and gel‐free proteomic approaches

The mitral valve is a highly complex structure which regulates blood flow from the left atrium to the left ventricle (LV) avoiding a significant forward gradient during diastole or regurgitation during systole. The integrity of the mitral valve is also essential for the maintenance of normal LV size, geometry, and function. Significant advances in the comprehension of the biological, functional, and mechanical behavior of the mitral valve have recently been made. However, current knowledge of protein components in the normal human mitral valve is still limited and complicated by the low cellularity of this tissue and the presence of high abundant proteins from the extracellular matrix. We employed here an integrated proteomic approach to analyse the protein composition of the normal human mitral valve and reported confident identification of 422 proteins, some of which have not been previously described in this tissue. In particular, we described the ability of pre‐MS separation technique based on liquid‐phase IEF and SDS‐PAGE to identify the largest number of proteins. We also demonstrated that some of these proteins, e.g. αB‐Crystallin, septin‐11, four‐and‐a‐half LIM domains protein 1, and dermatopontin, are synthesised by interstitial cells isolated from human mitral valves. These initial results provide a valuable basis for future studies aimed at analysing in depth the mitral valve protein composition and at investigating potential pathogenetic molecular mechanisms.