Elisa Bertacco

Widespread Increase in Myeloid Calcifying Cells Contributes to Ectopic Vascular Calcification in Type 2 Diabetes

Rationale: Acquisition of a procalcific phenotype by resident or circulating cells is important for calcification of atherosclerotic plaques, which is common in diabetes. Objective: We aim to identify and characterize circulating calcifying cells, and to delineate a pathophysiological role for these cells in type 2 diabetes. Methods and Results: We demonstrate for the first time that a distinct subpopulation of circulating cells expressing osteocalcin and bone alkaline phosphatase (OC+BAP+) has procalcific activity in vitro and in vivo. The study of naïve patients with chronic myeloid leukemia indicated that OC+BAP+ cells have a myeloid origin. Myeloid calcifying OC+BAP+ cells (MCCs) could be differentiated from peripheral blood mononuclear cells, and generation of MCCs was closely associated with expression of the osteogenic transcription factor Runx2. In gender-mismatched bone marrow–transplanted humans, circulating MCCs had a much longer half-life compared with OC−BAP− cells, suggesting they belong to a stable cell repertoire. The percentage of MCCs was higher in peripheral blood and bone marrow of type 2 diabetic patients compared with controls but was lowered toward normal levels by optimization of glycemic control. Furthermore, diabetic carotid endoarterectomy specimens showed higher degree of calcification and amounts of cells expressing OC and BAP in the &agr;-smooth muscle actin–negative areas surrounding calcified nodules, where CD68+ macrophages colocalize. High glucose increased calcification by MCCs in vitro, and hypoxia may regulate MCC generation in vitro and in vivo. Conclusions: These data identify a novel type of blood-derived procalcific cells potentially involved in atherosclerotic calcification of diabetic patients.

Hypertension and vascular calcification: a vicious cycle?

It is now well established that hypertension is accompanied by remodeling of the arterial wall with significant modifications in extracellular matrix composition and in vascular cell phenotype. Some of these changes, particularly elastin fragments generation, increased proteases activity and activation of transforming growth factor-&bgr; signaling together with deposition of collagen and proteoglycans might generate a permissive soil for vascular calcification. On the other hand, calcium deposits within large arterial conduits can reduce vessels elasticity and contribute to the generation of blood pressure pattern associated with vascular stiffness, namely isolated systolic hypertension. Hence, a hypothetical vicious cycle exists between hypertensive arterial damage and vascular calcification. Herein, we revised clinical and basic science findings supporting this possibility.

Clones of Interstitial Cells From Bovine Aortic Valve Exhibit Different Calcifying Potential When Exposed to Endotoxin and Phosphate

Objective—Our purpose was to study in vitro whether phenotypically-distinct interstitial cell clones from bovine aortic valve (BVIC) possess different calcifying potential in response to endotoxin (lipopolysaccharide [LPS]) and phosphate (Pi). Methods and Results—Among various clones of BVIC obtained by limited dilution technique we selected 4 clones displaying different growth patterns and immunophenotypes. Uncloned and cloned cells were treated with combinations of LPS (100 ng/mL) and Pi (2.4 mmol/L). Uncloned BVIC showed increased alkaline phosphatase activity (ALP) after treatment with LPS, which resulted in calcification after addition of Pi. Among BVIC clones, only Clone 1 (fibroblast-like phenotype) showed a relevant increase in ALP after LPS treatment in parallel with prevention of smooth muscle (SM) α-actin accumulation. No effect was observed in clonal cells harboring a more stable SM cell-like profile (Clone 4). None of the isolated clones calcified but mineralization was induced in the presence of LPS plus Pi when Clone 1 was cocultured with Clone 4 or after seeding on type I collagen sponges. Conclusion—Endotoxin and phosphate can act as valve calcification promoters by targeting specific fibroblast-like interstitial valve cells that possess a unique procalcific potential.

Proteomic analysis of clonal interstitial aortic valve cells acquiring a pro-calcific profile.

Calcific degeneration represents the most frequent aortic valve disease observed in industrialized countries. Our aim is to study modifications in the cytosolic and membrane protein profile of aortic interstitial valve cells (VIC) acquiring a pro-calcific phenotype. We studied a clonal population of bovine VIC that expresses bone-related proteins (such as alkaline phosphatase [ALP]) and calcifies a collagen matrix in response to endotoxin (LPS) treatment. A proteomic analysis was performed on proteins extracted from cells treated for 12 days with LPS (100 ng/mL) versus control. We identified 34 unique cytosolic and 10 unique membrane-associated proteins showing significant changes after treatment. These proteins are involved in several cellular functions, such as chaperone-mediated protein folding, protein metabolism and transport, cell redox/nitric oxide homeostasis, and cytoskeletal organization. Reduced expression of proteins involved in NOS bioactivity (such as DDAH-1 and -2) suggested a role for the l-arginine/ADMA ratio in controlling VIC phenotypic profile. In accordance with this hypothesis, we observed that exposure of clonal cells to l-arginine prevented LPS-induced ALP expression and collagen calcification. In conclusion, we identified several proteins involved in structural, metabolic, and signaling functions that are significantly altered in aortic VIC acquiring a pro-calcific profile, thus giving new insights into the pathogenesis of aortic valve degeneration.

Aortic valve calcification in chronic kidney disease

Several clinical studies reported an increased prevalence and accelerated progression of aortic valve calcification among patients with end-stage renal disease when compared with subjects with normal kidney function. Recently, mechanisms of calcific valve degeneration have been further elucidated and many of the pathways involved could be amplified in patients with decreased renal function. In particular, calcium-phosphate balance, MGP metabolism, OPG/RANK/RANKL triad, fetuin-A mineral complexes and FGF-23/Klotho axis have been shown to be impaired among patients with advanced chronic kidney disease and could play a role during vascular/valve calcification. The scope of the present review is to summarize the clinical data and the pathophysiological mechanisms potentially involved in the link between renal function decline and the progression of aortic valve disease.

Delta2D and Proteomweaver: Performance evaluation of two different approaches for 2-DE analysis

2‐DE is a fundamental technology used in proteomics research. However, despite its high capacity to simultaneously separate several proteins for subsequent identification and quantitative comparison studies, a drawback for this technique is its limited reproducibility, especially when comparing data from different laboratories. 2‐DE‐related variability can be broadly divided into two categories: experimental and post‐experimental. Experimental variability depends on physical and chemical parameters, whereas post‐experimental variability arises when gels are analyzed by different software packages, particularly when different workflows are followed. In this paper, we compared the analysis performance of two software packages, Delta2D and Proteomweaver, using both standard and experimental gel images. Using standard gel images, the false negative spot count was 50% lower, the false positive count was 77% lower, the true positive count was 19% higher and spot matching was 4% higher in Delta2D when compared to Proteomeweaver. Using experimental gel images, we found that the total amount of time taken to complete the analysis with Delta2D was 30% that of the time needed with Proteomweaver and required fewer user interventions. The differences between ease of use and workflow strategy of these programs is discussed.

Extracellular pyrophosphate is reduced in aortic interstitial valve cells acquiring a calcifying profile: Implications for aortic valve calcification

OBJECTIVES Pyrophosphate (PPi) is a potent inhibitor of ectopic mineralization but its role during aortic valve calcification is not known. METHODS Anti-calcific effect of PPi was investigated by using an in vitro model of serum-driven calcification of collagen sponges and decellularized porcine aortic valve leaflets. Bovine interstitial valve cells (VIC), seeded either within the collagen matrices or in transwell chambers, were used to test cellular ability to inhibit serum-induced calcification. PPi metabolism was investigated in clonal VIC harboring different calcifying potential. RESULTS In a cell-free system, high serum levels induced a dose-dependent calcification of type I collagen matrices which was prevented by PPi and ATP supplementation. Blockade of serum-driven calcification by PPi and ATP was also observed when using decellularized porcine aortic valve leaflets. A similar anti-calcific effect was also seen for bovine VIC, either statically seeded into the collagen matrices or co-cultured by using a transwell system. However, when we performed co-culture experiments by using clonal VIC harboring different calcifying potential, we observed that the subset of cells acquiring a pro-calcific profile lost the ability to protect the collagen from serum-driven calcification. Pro-calcific differentiation of the clonal VIC was accompanied by increase in ALP along with significant reduction in NPP activity and ATP/PPi extracellular accumulation. These changes were not observed in the clonal subtype with lower propensity towards calcification. CONCLUSIONS We showed that PPi and ATP are potent inhibitors of serum-driven calcification of collagen matrix and that their extracellular accumulation is reduced in calcifying VIC.

Low CD34(+) cells, high neutrophils and the metabolic syndrome are associated with an increased risk of venous thromboembolism.

The relationship between MetS (metabolic syndrome), levels of circulating progenitor/immune cells and the risk of VTE (venous thromboembolism) has not yet been investigated. We studied 240 patients with previous VTE and 240 controls. The presence of MetS was identified according to NCEP ATP III guidelines and flow cytometry was used to quantify circulating CD34(+) cells. VTE patients showed higher BMI (body mass index), waist circumference, triacylglycerol (triglyceride) levels, blood glucose, hs-CRP (high-sensitivity C-reactive protein) and lower HDL-C (high-density lipoprotein cholesterol) levels. The prevalence of MetS was significantly higher in VTE (38.3%) than in control individuals (21.3%) with an adjusted OR (odds ratio) for VTE of 1.96 (P=0.002). VTE patients had higher circulating neutrophils (P<0.0001), while the CD34(+) cell count was significantly lower among patients with unprovoked VTE compared with both provoked VTE (P=0.004) and controls (P=0.003). Subjects were also grouped according to the presence/absence of MetS (MetS(+) or MetS(-)) and the level (high/low) of both CD34(+) cells and neutrophils. Very high adjusted ORs for VTE were observed among neutrophils_high/MetS(+) (OR, 3.58; P<0.0001) and CD34(+)_low/MetS(+) (OR, 3.98; P<0.0001) subjects as compared with the neutrophils_low/MetS(-) and CD34(+)_high/MetS(-) groups respectively. In conclusion, low CD34(+) blood cell count and high circulating neutrophils interplay with MetS in raising the risk for venous thromboembolic events.

RANKL Expression Is Increased in Circulating Mononuclear Cells of Patients with Calcific Aortic Stenosis

We aimed to investigate whether the expression of the OPG/RANK/RANKL triad in peripheral blood mononuclear cells (PBMC) and circulating levels of markers of ectopic mineralization (OPG, FGF-23, PPi) are modified in patients with calcific aortic valve disease (CAVD). We found that patients affected by CAVD (n = 50) had significantly higher circulating levels of OPG as compared to control individuals (p = 0.003). No differences between the two groups were found in FGF-23 and PPi levels. RANKL expression was higher in the PBMC from CAVD patients (p = 0.018) and was directly correlated with the amount of valve calcification (p = 0.032). In vitro studies showed that treatment of valve interstitial cells (VIC) with RANKL plus phosphate was followed by increase in matrix mineralization (p = 0.001). In conclusion, RANKL expression is increased in PBMC of patients with CAVD, is directly correlated with the degree of valve calcification, and promotes pro-calcific differentiation of VIC.