Ca2+ dysregulation in cardiac stromal cells sustains fibro-adipose remodeling in Arrhythmogenic Cardiomyopathy and can be modulated by flecainide.

BACKGROUND: Cardiac mesenchymal stromal cells (C-MSC) were recently shown to differentiate into adipocytes and myofibroblasts to promote the aberrant remodeling of cardiac tissue that characterizes arrhythmogenic cardiomyopathy (ACM). A calcium (Ca2+) signaling dysfunction, mainly demonstrated in mouse models, is recognized as a mechanism impacting arrhythmic risk in ACM cardiomyocytes. Whether similar mechanisms influence ACM C-MSC fate is still unknown. Thus, we aim to ascertain whether intracellular Ca2+ oscillations and the Ca2+ toolkit are altered in human C-MSC obtained from ACM patients, and to assess their link with C-MSC-specific ACM phenotypes. METHODS AND RESULTS: ACM C-MSC show enhanced spontaneous Ca2+ oscillations and concomitant increased Ca2+/Calmodulin dependent kinase II (CaMKII) activation compared to control cells. This is manly linked to a constitutive activation of Store-Operated Ca2+ Entry (SOCE), which leads to enhanced Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors. By targeting the Ca2+ handling machinery or CaMKII activity, we demonstrated a causative link between Ca2+ oscillations and fibro-adipogenic differentiation of ACM C-MSC. Genetic silencing of the desmosomal gene PKP2 mimics the remodelling of the Ca2+ signalling machinery occurring in ACM C-MSC. The anti-arrhythmic drug flecainide inhibits intracellular Ca2+ oscillations and fibro-adipogenic differentiation by selectively targeting SOCE. CONCLUSIONS: Altogether, our results extend the knowledge of Ca2+ dysregulation in ACM to the stromal compartment, as an etiologic mechanism of C-MSC-related ACM phenotypes. A new mode of action of flecainide on a novel mechanistic target is unveiled against the fibro-adipose accumulation in ACM.

N-Acetylcysteine Regenerates In Vivo Mercaptoalbumin.

Human serum albumin (HSA) represents the most abundant plasma protein, with relevant antioxidant activity due to the presence of the sulfhydryl group on cysteine at position 34 (Cys34), the latter being one of the major target sites for redox-dependent modifications leading to the formation of mixed disulfide linkages with low molecular weight thiols. Thiolated forms of HSA (Thio-HSA) may be useful as markers of an unbalanced redox state and as a potential therapeutic target. Indeed, we have previously reported that albumin Cys34 can be regenerated in vitro by N-Acetylcysteine (NAC) through a thiol-disulfide breaking mechanism, with a full recovery of the HSA antioxidant and antiplatelet activities. With this case study, we aimed to assess the ability of NAC to regenerate native mercaptoalbumin (HSA-SH) and the plasma antioxidant capacity in subjects with redox unbalance, after oral and intravenous administration. A placebo-controlled crossover study, single-blinded, was performed on six hypertensive subjects, randomized into two groups, on a one-to-one basis with NAC (600 mg/die) or a placebo, orally and intravenously administered. Albumin isoforms, HSA-SH, Thio-HSA, and glutathione levels were evaluated by means of mass spectrometry. The plasma antioxidant activity was assessed by a fluorimetric assay. NAC, orally administered, significantly decreased the Thio-HSA levels in comparison with the pre-treatment conditions (T0), reaching the maximal effect after 60 min (-24.7 ± 8%). The Thio-HSA reduction was accompanied by a concomitant increase in the native HSA-SH levels (+6.4 ± 2%). After intravenous administration of NAC, a significant decrease of the Thio-HSA with respect to the pre-treatment conditions (T0) was observed, with a maximal effect after 30 min (-68.9 ± 10.6%) and remaining significant even after 6 h. Conversely, no effect on the albumin isoforms was detected with either the orally or the intravenously administered placebo treatments. Furthermore, the total antioxidant activity of the plasma significantly increased after NAC infusion with respect to the placebo (p = 0.0089). Interestingly, we did not observe any difference in terms of total glutathione corrected for hemoglobin, ruling out any effect of NAC on the intracellular glutathione and supporting its role as a disulfide-breaking agent. This case study confirms the in vitro experiments and demonstrates for the first time that NAC is able to regenerate mercaptoalbumin in vivo, allowing us to hypothesize that the recovery of Cys34 content can modulate in vivo oxidative stress and, hopefully, have an effect in oxidative-based diseases.

Pressure Overload Activates DNA-Damage Response in Cardiac Stromal Cells: A Novel Mechanism Behind Heart Failure With Preserved Ejection Fraction?

Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome characterized by impaired left ventricular (LV) diastolic function, with normal LV ejection fraction. Aortic valve stenosis can cause an HFpEF-like syndrome by inducing sustained pressure overload (PO) and cardiac remodeling, as cardiomyocyte (CM) hypertrophy and fibrotic matrix deposition. Recently, in vivo studies linked PO maladaptive myocardial changes and DNA damage response (DDR) activation: DDR-persistent activation contributes to mouse CM hypertrophy and inflammation, promoting tissue remodeling, and HF. Despite the wide acknowledgment of the pivotal role of the stromal compartment in the fibrotic response to PO, the possible effects of DDR-persistent activation in cardiac stromal cell (C-MSC) are still unknown. Finally, this novel mechanism was not verified in human samples. This study aims to unravel the effects of PO-induced DDR on human C-MSC phenotypes. Human LV septum samples collected from severe aortic stenosis with HFpEF-like syndrome patients undergoing aortic valve surgery and healthy controls (HCs) were used both for histological tissue analyses and C-MSC isolation. PO-induced mechanical stimuli were simulated in vitro by cyclic unidirectional stretch. Interestingly, HFpEF tissue samples revealed DNA damage both in CM and C-MSC. DDR-activation markers γH2AX, pCHK1, and pCHK2 were expressed at higher levels in HFpEF total tissue than in HC. Primary C-MSC isolated from HFpEF and HC subjects and expanded in vitro confirmed the increased γH2AX and phosphorylated checkpoint protein expression, suggesting a persistent DDR response, in parallel with a higher expression of pro-fibrotic and pro-inflammatory factors respect to HC cells, hinting to a DDR-driven remodeling of HFpEF C-MSC. Pressure overload was simulated in vitro, and persistent activation of the CHK1 axis was induced in response to in vitro mechanical stretching, which also increased C-MSC secreted pro-inflammatory and pro-fibrotic molecules. Finally, fibrosis markers were reverted by the treatment with a CHK1/ATR pathway inhibitor, confirming a cause-effect relationship. In conclusion we demonstrated that, in severe aortic stenosis with HFpEF-like syndrome patients, PO induces DDR-persistent activation not only in CM but also in C-MSC. In C-MSC, DDR activation leads to inflammation and fibrosis, which can be prevented by specific DDR targeting.

Apolipoprotein Signature of HDL and LDL from Atherosclerotic Patients in Relation with Carotid Plaque Typology: A Preliminary Report.

In the past years, it has become increasingly clear that the protein cargo of the different lipoprotein classes is largely responsible for carrying out their various functions, also in relation to pathological conditions, including atherosclerosis. Accordingly, detailed information about their apolipoprotein composition and structure may contribute to the revelation of their role in atherogenesis and the understanding of the mechanisms that lead to atherosclerotic degeneration and toward vulnerable plaque formation. With this aim, shotgun proteomics was applied to identify the apolipoprotein signatures of both high-density and low-density lipoproteins (HDL and LDL) plasma fractions purified from healthy volunteers and atherosclerotic patients with different plaque typologies who underwent carotid endarterectomy. By this approach, two proteins with potential implications in inflammatory, immune, and hemostatic pathways, namely, integrin beta-2 (P05107) and secretoglobin family 3A member 2 (Q96PL1), have been confirmed to belong to the HDL proteome. Similarly, the list of LDL-associated proteins has been enriched with 21 proteins involved in complement and coagulation cascades and the acute-phase response, which potentially double the protein species of LDL cargo. Moreover, differential expression analysis has shown protein signatures specific for patients with "hard" or "soft" plaques.

Cardiovascular tissue banking activity during SARS-CoV-2 pandemic: evolution of national protocols and Lombardy experience.

The worldwide pandemic outbreak due to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has created unprecedented challenges for public health services. Lombardy, region of the Northern Italy, has been the first area in the Western world whose organs and tissues procurement programs have had to face the virus pandemic emergency. We retrospectively collected and analyzed data about cardiovascular tissues (CT) in 2019 and in 2020. We aimed to describe the rapid evolution of SARS-CoV-2 regulation laws for tissue donor's selection and harvesting from February 2020 until January 2021. As expected the number of CT donors in 2020 was significantly lower than those of 2019 (66 vs. 99, p value 0.02). The total number of CT collected from donors have been 254 in 2019 and 206 in 2020 (p 0.28). Femoral arteries were the most required vascular tissues (55.5% in 2019 and 40% in 2020). Fifty-five and forty-eight pulmonary valves were implanted in 2019 and 2020, respectively. No differences were found for the types of CT requests between the 2 years. The median age of receivers of vascular tissues was 69.6 ± 14.6 years in the 2019 and 63.3 ± 14.9 years in 2020 (p < 0.01). The median age of receivers of pulmonary and aortic valves did not differ between the 2 years (9.32 ± 11.49 vs. 8.36 ± 10.66 and 48.67 ± 27.19 vs. 37.14 ± 31.97 respectively). Despite the dramatically reduction of donors, the number of CT collected has not decreased significantly and so far the CT distribution rate is comparable to those of 2019.

Neuropeptide Y promotes adipogenesis of human cardiac mesenchymal stromal cells in arrhythmogenic cardiomyopathy.

BACKGROUND: Arrhythmogenic Cardiomyopathy (AC) is a familial cardiac disease, mainly caused by mutations in desmosomal genes. AC hearts show fibro-fatty myocardial replacement, which favors stress-related life-threatening arrhythmias, predominantly in the young and athletes. AC lacks effective therapies, as its pathogenesis is poorly understood. Recently, we showed that cardiac Mesenchymal Stromal Cells (cMSCs) contribute to adipose tissue in human AC hearts, although the underlying mechanisms are still unclear. PURPOSE: We hypothesize that the sympathetic neurotransmitter, Neuropeptide Y (NPY), participates to cMSC adipogenesis in human AC. METHODS: For translation of our findings, we combined in vitro cytochemical, molecular and pharmacologic assays on human cMSCs, from myocardial biopsies of healthy controls and AC patients, with the use of existing drugs to interfere with the predicted AC mechanisms. Sympathetic innervation was inspected in human autoptic heart samples, and NPY plasma levels measured in healthy and AC subjects. RESULTS: AC cMSCs expressed higher levels of pro-adipogenic isotypes of NPY-receptors (i.e. Y1-R, Y5-R). Consistently, NPY enhanced adipogenesis in AC cMSCs, which was blocked by FDA-approved Y1-R and Y5-R antagonists. AC-associated PKP2 reduction directly caused NPY-dependent adipogenesis in cMSCs. In support of the involvement of sympathetic neurons (SNs) and NPY in AC myocardial remodeling, patients had elevated NPY plasma levels and, in human AC hearts, SNs accumulated in fatty areas and were close to cMSCs. CONCLUSIONS: Independently from the disease origin, AC causes in cMSCs a targetable gain of responsiveness to NPY, which leads to increased adipogenesis, thus playing a role in AC myocardial remodeling.

Excess TGF-ß1 Drives Cardiac Mesenchymal Stromal Cells to a Pro-Fibrotic Commitment in Arrhythmogenic Cardiomyopathy.

Arrhythmogenic Cardiomyopathy (ACM) is characterized by the replacement of the myocardium with fibrotic or fibro-fatty tissue and inflammatory infiltrates in the heart. To date, while ACM adipogenesis is a well-investigated differentiation program, ACM-related fibrosis remains a scientific gap of knowledge. In this study, we analyze the fibrotic process occurring during ACM pathogenesis focusing on the role of cardiac mesenchymal stromal cells (C-MSC) as a source of myofibroblasts. We performed the ex vivo studies on plasma and right ventricular endomyocardial bioptic samples collected from ACM patients and healthy control donors (HC). In vitro studies were performed on C-MSC isolated from endomyocardial biopsies of both groups. Our results revealed that circulating TGF-ß1 levels are significantly higher in the ACM cohort than in HC. Accordingly, fibrotic markers are increased in ACM patient-derived cardiac biopsies compared to HC ones. This difference is not evident in isolated C-MSC. Nevertheless, ACM C-MSC are more responsive than HC ones to TGF-ß1 treatment, in terms of pro-fibrotic differentiation and higher activation of the SMAD2/3 signaling pathway. These results provide the novel evidence that C-MSC are a source of myofibroblasts and participate in ACM fibrotic remodeling, being highly responsive to ACM-characteristic excess TGF-ß1.

Presence of SARS-CoV-2 Nucleoprotein in Cardiac Tissues of Donors with Negative COVID-19 Molecular Tests.

The 2019 Coronavirus disease (COVID-19) outbreak had detrimental effects on essential medical services such as organ and tissue donation. Lombardy, one of the most active Italian regions in organ/tissue procurement, has been strongly affected by the COVID-19 pandemic. To date, data concerning the risk of SARS-CoV-2 transmission after tissue transplantation are controversial. Here, we aimed to evaluate the presence/absence of SARS-CoV-2 in different cardiac tissues eligible for transplantation obtained from Lombard donors. We used cardiovascular tissues from eight donors potentially suitable for pulmonary valve transplantation. All donor subjects involved in the study returned negative results for the SARS-CoV-2 RNA molecular tests (quantitative real-time reverse-transcription PCR, qRT-PCR, and chip-based digital PCR) in nasopharyngeal swabs (NPS) or bronchoalveolar lavage (BAL). None of the eight donors included in this study revealed the presence of the SARS-CoV-2 viral genome. However, evaluation of the protein content of pulmonary vein wall (PVW) tissue revealed variable levels of SARS-CoV-2 nucleoprotein signal in all donors. Our study demonstrated for the first time, to the best of our knowledge, that viral nucleoprotein but not viral RNA was present in the examined tissue bank specimens, suggesting the need for caution and in-depth investigations on implantable tissue specimens collected during the COVID-19 pandemic period.

Human Cardiac Mesenchymal Stromal Cells From Right and Left Ventricles Display Differences in Number, Function, and Transcriptomic Profile.

BACKGROUND: Left ventricle (LV) and right ventricle (RV) are characterized by well-known physiological differences, mainly related to their different embryological origin, hemodynamic environment, function, structure, and cellular composition. Nevertheless, scarce information is available about cellular peculiarities between left and right ventricular chambers in physiological and pathological contexts. Cardiac mesenchymal stromal cells (C-MSC) are key cells affecting many functions of the heart. Differential features that distinguish LV from RV C-MSC are still underappreciated. AIM: To analyze the physiological differential amount, function, and transcriptome of human C-MSC in LV versus (vs.) RV. METHODS: Human cardiac specimens of LV and RV from healthy donors were used for tissue analysis of C-MSC number, and for C-MSC isolation. Paired LV and RV C-MSC were compared as for surface marker expression, cell proliferation/death ratio, migration, differentiation capabilities, and transcriptome profile. RESULTS: Histological analysis showed a greater percentage of C-MSC in RV vs. LV tissue. Moreover, a higher C-MSC amount was obtained from RV than from LV after isolation procedures. LV and RV C-MSC are characterized by a similar proportion of surface markers. Functional studies revealed comparable cell growth curves in cells from both ventricles. Conversely, LV C-MSC displayed a higher apoptosis rate and RV C-MSC were characterized by a higher migration speed and collagen deposition. Consistently, transcriptome analysis showed that genes related to apoptosis regulation or extracellular matrix organization and integrins were over-expressed in LV and RV, respectively. Besides, we revealed additional pathways specifically associated with LV or RV C-MSC, including energy metabolism, inflammatory response, cardiac conduction, and pluripotency. CONCLUSION: Taken together, these results contribute to the functional characterization of RV and LV C-MSC in physiological conditions. This information suggests a possible differential role of the stromal compartment in chamber-specific pathologic scenarios.

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. Data are available via ProteomeXchange with identifier PXD004397.

Identification of differentially expressed plasma proteins in atherosclerotic patients with type 2 diabetes.

Besides hyperglycaemia and insulin resistance, several factors are associated with a higher cardiovascular risk in type 2 diabetes mellitus (T2DM), many of them being closely related to each other owing to common origins or pathways. The pathophysiological mechanisms underlying vascular dysfunctions in diabetes include reduced bioavailability of nitric oxide, increased ROS and prothrombotic factors production, as well as activation of receptors for advanced glycation end-products. These alterations contribute to create a pro-inflammatory/thrombotic state that ultimately leads to plaque formation and complication. This study aimed at identifying differentially expressed plasma proteins between T2DM and non-diabetic patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis coupled with LC-MS/MS. Before analysis, plasma samples were enriched in low-expression proteins through combinatorial hexapeptide ligand libraries. Both mono- and two-dimensional western blotting were performed for data validation. Differentially expressed proteins were mapped onto STRING v10 to build a protein-protein interaction network. Sixteen differentially expressed spots were identified with a high score. Among them, there were fibrinogen beta and gamma chains, complement C1r, C3 and C4-B subcomponents, alpha-1-antitrypsin (AAT), vitronectin and CD5 antigen-like. Protein-Protein interaction analysis evidenced a network among differentially expressed proteins in which vitronectin seems to represent a potentially pivotal node among fibrinolysis, complement dependent immune responses and inflammation in accordance with a number of in vitro and in vivo evidences for a contributory role of these proteins to the development of diabetic atherosclerosis.

Residual antibiotics in decontaminated human cardiovascular tissues intended for transplantation and risk of falsely negative microbiological analyses.

We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.

Human serum albumin Cys34 oxidative modifications following infiltration in the carotid atherosclerotic plaque.

OBJECTIVES: To evaluate if the prooxidant environment present in atherosclerotic plaque may oxidatively modify filtered albumin. METHODS: Fluorescein-5-maleimide labelled plasma samples and plaque extracts from 27 patients who had undergone carotid endarterectomy were analysed through nonreducing SDS-PAGE for albumin-Cys(34) oxidation. Furthermore, degree and pattern of S-thiolation in both circulating and plaque-filtered albumin were assayed. RESULTS: Albumin filtered in the atherosclerotic plaque showed higher levels of Cys(34) oxidative modifications than the corresponding circulating form as well as different patterns of S-thiolation. CONCLUSIONS: Data indicate that the circulating albumin, once filtered in plaque, undergoes Cys(34) oxidative modifications and demonstrate for the first time that albumin is a homocysteine and cysteinylglycine vehicle inside the plaque environment.

Aortic valve leaflet glycosaminoglycans composition and modification in severe chronic valve regurgitation.

BACKGROUND AND AIM OF THE STUDY: The surgical segments of aortic valve leaflets from patients with severe chronic aortic regurgitation were analyzed (by percentage and structure) for their content of complex polysaccharides and glycosaminoglycans (GAGs), and compared with control segments. METHODS: The GAG, hyaluronic acid (HA), chondroitin sulfate (CS) and dermatan sulfate (DS) and disaccharide contents were determined in segments (leaflet, root attachment region and belly) of aortic valve leaflets (non-coronary, left coronary and right coronary) using a multi-analytical approach. RESULTS: The aortic valve leaflets showed the presence of HA and CS/DS, with an overall charge density of -0.51-0.55. The CS/DS polymers showed a 4-sulfated/6-sulfated ratio of -0.70-0.77 in the belly, and -1.60-1.72 in commissure parts (-/+124%). The total amount of GAGs was -1.60-2.40 microg/mg of tissue. A significant increase in sulfated GAGs was observed in all valve parts in patients suffering from severe aortic insufficiency, as well as an increase in the 4-sulfated/6-sulfated ratio in the leaflet belly (-/+102%). CONCLUSION: It is speculated that differences in 4-sulfated/6-sulfated ratio determined in the belly and leaflet attachment region-commissure parts of the leaflets may correlate with the tensile or compressive loading of normal aortic valve regions. At the same time, it may be assumed that the increase in sulfated GAGs and 4-sulfated/6-sulfated ratio in the leaflet belly of valves taken from patients suffering severe aortic insufficiency was consistent with an altered matrix microstructure capable of influencing the hydration of these pathological tissues, and of conditioning their mechanical weakness.

Protein sulfhydryl group oxidation and mixed-disulfide modifications in stable and unstable human carotid plaques.

OBJECTIVES: Oxidative stress has been implicated in the outcome of atherosclerotic plaques. However, at present, no data are available neither on the degree of plaque protein sulfhydryl groups oxidation nor on its relationship with plaque vulnerability. We investigated the entity of protein-SH oxidative modifications, focusing on low molecular weight thiols adduction, in human carotid plaque extracts in relation to plaque stability/instability. METHODS: Plaque stability/instability was histologically assessed. The extent of protein-SH oxidative modifications was established by a differential proteomic approach on fluorescein-5-maleimide-labeled plaque extracts and corresponding plasma samples from 48 endarterectomized patients. The analysis on protein thiolation was performed by capillary zone electrophoresis. RESULTS: We observed a higher protein-SH oxidation of both plasma-derived and topically expressed proteins in unstable plaques, partly due to higher levels of S-thiolation. Conversely, in plasma, none of the investigated parameters discriminated among patients with stable and unstable plaques. CONCLUSIONS: Our results suggest the presence of a more pronounced oxidative environment in unstable plaques. Identifying specific oxidative modifications and understanding their effects on protein function could provide further insight into the relevance of oxidative stress in atherosclerosis.

Establishing a procedure for dimethyl sulfoxide removal from cardiovascular allografts: a quantitative study.

The success of allotransplants is critically dependent on both tissue viability and efficient removal of potentially toxic cryopreservants. In this study we analysed the dimethyl sulphoxide (Me2SO) content of cardiovascular tissue samples stored in tissue banks and optimized a washing protocol to be used before surgical implant. We compared the Me2SO content of heart valves, arteries and veins and quantitatively determined by HPLC the washing kinetics of each group of tissue samples under strictly controlled conditions using an industrial washing medium (BASE). Our results showed that heart valves and arteries have significantly slower Me2SO release kinetics than veins. Approximately 20 % of the initial content of cryopreservant could still be detected in the valves and arterial tissue after 15 min of continuous washing. Conversely, veins were almost completely cleared of the cryoprotectant under the same conditions. We propose a washing protocol consisting of two sequential washing with BASE for a total of 25 min for valves and arteries and 15 min for veins. In our hands, this protocol reliably ensures the removal of more than 95 % of the initial Me2SO content.

Proteomic analysis of plasma-purified VLDL, LDL, and HDL fractions from atherosclerotic patients undergoing carotid endarterectomy: identification of serum amyloid A as a potential marker.

Apolipoproteins are very heterogeneous protein family, implicated in plasma lipoprotein structural stabilization, lipid metabolism, inflammation, or immunity. Obtaining detailed information on apolipoprotein composition and structure may contribute to elucidating lipoprotein roles in atherogenesis and to developing new therapeutic strategies for the treatment of lipoprotein-associated disorders. This study aimed at developing a comprehensive method for characterizing the apolipoprotein component of plasma VLDL, LDL, and HDL fractions from patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis (2-DE) coupled with Mass Spectrometry analysis, useful for identifying potential markers of plaque presence and vulnerability. The adopted method allowed obtaining reproducible 2-DE maps of exchangeable apolipoproteins from VLDL, LDL, and HDL. Twenty-three protein isoforms were identified by peptide mass fingerprinting analysis. Differential proteomic analysis allowed for identifying increased levels of acute-phase serum amyloid A protein (AP SAA) in all lipoprotein fractions, especially in LDL from atherosclerotic patients. Results have been confirmed by western blotting analysis on each lipoprotein fraction using apo AI levels for data normalization. The higher levels of AP SAA found in patients suggest a role of LDL as AP SAA carrier into the subendothelial space of artery wall, where AP SAA accumulates and may exert noxious effects.

Heart valve engineering: decellularized aortic homograft seeded with human cardiac stromal cells.

BACKGROUND AND AIM OF THE STUDY: The adult human heart contains a cardiac mesenchymal stromal cell (CStC) population with residual cardiovascular plasticity. The study aim was to investigate the ability of CStCs to populate decellularized aortic homograft leaflets, without mechanical stimulation. METHODS: The ability of CStCs to acquire valve endothelial and interstitial cell phenotypes was tested using in vitro assays. First, trypsin-decellularized aortic leaflets were seeded with CStCs under static conditions; tissue section analyses were then performed before and after decellularization, and at 10, 20, and 30 days after reseeding. RESULTS: Following in vitro treatment, the CStCs differentiated along the endothelial lineage, as shown by their capacity to uptake acetylated low-density lipoprotein and to secrete the pro-angiogenic factor, vascular endothelial growth factor. After seeding, CStCs were able to adhere to the leaflet surface, rescuing up to the 90% of the original cell density and expressing the mature endothelial marker, von Willebrandt factor. The CStC supernatants were also positive for matrix metalloprotease-2 (MMP-2), which confirmed the ability of these cells to penetrate within the leaflet structure; this also suggested that CStCs, once engrafted, would contribute to the extracellular matrix turnover. Accordingly, although at a lower efficiency, CStC repopulation was also evident in the inner portions of the leaflet. CONCLUSION: Seeded CStCs were able to reconstitute, without mechanical stimulation, an endothelial-like layer and to partially infiltrate decellularized homograft leaflets. Hence, CStCs appear to be a potentially useful cell type for engineered heart valves.

Association between Human Plasma Chondroitin Sulfate Isomers and Carotid Atherosclerotic Plaques.

Several studies have evidenced variations in plasma glycosaminoglycans content in physiological and pathological conditions. In normal human plasma GAGs are present mainly as undersulfated chondroitin sulfate (CS). The aim of the present study was to evaluate possible correlations between plasma CS level/structure and the presence/typology of carotid atherosclerotic lesion. Plasma CS was purified from 46 control subjects and 47 patients undergoing carotid endarterectomy showing either a soft or a hard plaque. The concentration and structural characteristics of plasma CS were assessed by capillary electrophoresis of constituent unsaturated fluorophore-labeled disaccharides. Results showed that the concentration of total CS isomers was increased by 21.4% (P < 0.01) in plasma of patients, due to a significant increase of undersulfated CS. Consequently, in patients the plasma CS charge density was significantly reduced with respect to that of controls. After sorting for plaque typology, we found that patients with soft plaques and those with hard ones differently contribute to the observed changes. In plasma from patients with soft plaques, the increase in CS content was not associated with modifications of its sulfation pattern. On the contrary, the presence of hard plaques was associated with CS sulfation pattern modifications in presence of quite normal total CS isomers levels. These results suggest that the plasma CS content and structure could be related to the presence and the typology of atherosclerotic plaque and could provide a useful diagnostic tool, as well as information on the molecular mechanisms responsible for plaque instability.