Inflammatory and Biomechanical Drivers of Endothelial-Interstitial Interactions in Calcific Aortic Valve Disease.

Calcific aortic valve disease is dramatically increasing in global burden, yet no therapy exists outside of prosthetic replacement. The increasing proportion of younger and more active patients mandates alternative therapies. Studies suggest a window of opportunity for biologically based diagnostics and therapeutics to alleviate or delay calcific aortic valve disease progression. Advancement, however, has been hampered by limited understanding of the complex mechanisms driving calcific aortic valve disease initiation and progression towards clinically relevant interventions.

Simvastatin reduces the TLR4-induced inflammatory response in human aortic valve interstitial cells.

BACKGROUND: Calcific aortic stenosis is a chronic inflammatory disease. Proinflammatory stimulation via toll-like receptor 4 (TLR4) causes the aortic valve interstitial cell (AVIC) to undergo phenotypic change. The AVIC first assumes an inflammatory phenotype characterized by the production of inflammatory mediators such as intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). This change has been linked with an osteogenic phenotypic response. Statins have recently been shown to have anti-inflammatory properties. We therefore hypothesized that statins may have an anti-inflammatory effect on human AVICs by downregulation of TLR4-stimulated inflammatory responses. Our purposes were (1) to determine the effect of simvastatin on TLR4-induced expression of inflammatory mediators in human AVICs and (2) to determine the mechanism(s) through which simvastatin exert this effect. MATERIALS AND METHODS: Human AVICs were isolated from the explanted hearts of four patients undergoing cardiac transplantation. Cells were treated with simvastatin (50 µM) for 1 h before stimulation with TLR4 agonist lipopolysaccharide (LPS, 0.2 µg/mL). Immunoblotting (IB) was used to analyze cell lysates for ICAM-1 expression, and enzyme-linked immunosorbent assay was used to detect IL-8 and MCP-1 in cell culture media. Likewise, lysates were analyzed for TLR4 and nuclear factor-kappa B activation (IB). After simvastatin treatment, lysates were analyzed for TLR4 levels (IB). Statistics were by analysis of variance (P < 0.05). RESULTS: Simvastatin reduced TLR4-induced ICAM-1, IL-8, and MCP-1 expression in AVICs. Simvastatin down-regulated TLR4 levels and suppressed TLR4-induced phosphorylation of nuclear factor-kappa B. CONCLUSIONS: These data demonstrate the potential of a medical therapy (simvastatin) to impact the pathogenesis of aortic stenosis.

Survival-Related Autophagic Activity Versus Procalcific Death in Cultured Aortic Valve Interstitial Cells Treated With Critical Normophosphatemic-Like Phosphate Concentrations.

Valve dystrophic calcification is a common disorder affecting normophosphatemic subjects. Here, cultured aortic valve interstitial cells (AVICs) were treated 3 to 28 days with phosphate (Pi) concentrations spanning the normal range in humans (0.8, 1.3, and 2.0 mM) alone or supplemented with proinflammatory stimuli to assess possible priming of dystrophic-like calcification. Compared with controls, spectrophotometric analyses revealed marked increases in calcium amounts and alkaline phosphatase activity for 2.0-mM-Pi-containing cultures, with enhancing by proinflammatory mediators. Ultrastructurally, AVICs treated with low/middle Pi concentrations showed an enormous endoplasmic reticulum (ER) enclosing organelle debris, so apparently executing a survival-related atypical macroautophagocytosis, consistently with ultracytochemical demonstration of ER-associated acid phosphatase activity and decreases in autophagosomes and immunodetectable MAP1LC3. In contrast, AVICs cultured at 2.0-mM Pi underwent mineralization due to intracellular release and peripheral layering of phospholipid-rich material acting as hydroxyapatite nucleator, as revealed by Cuprolinic Blue and von Kossa ultracytochemical reactions. Lack of immunoblotted caspase-3 cleaved form indicated apoptosis absence for all cultures. In conclusion, fates of cultured AVICs were crucially driven by Pi concentration, suggesting that serum Pi levels just below the upper limit of normophosphatemia in humans may represent a critical watershed between macroautophagy-associated cell restoring and procalcific cell death.

Ascorbic acid promotes extracellular matrix deposition while preserving valve interstitial cell quiescence within 3D hydrogel scaffolds.

Current options for aortic valve replacements are non-viable and thus lack the ability to grow and remodel, which can be problematic for paediatric applications. Toward the development of living valve substitutes that can grow and remodel, porcine aortic valve interstitial cells (VICs) were isolated and encapsulated within proteolytically degradable and cell-adhesive poly(ethylene glycol) (PEG) hydrogels, in an effort to study their phenotypes and functions. The results showed that encapsulated VICs maintained high viability and proliferated within the hydrogels. The VICs actively remodelled the hydrogels via secretion of matrix metalloproteinase-2 (MMP-2) and deposition of new extracellular matrix (ECM) components, including collagens I and III. The soft hydrogels with compressive moduli of ~4.3 kPa quickly reverted VICs from an activated myofibroblastic phenotype to a quiescent, unactivated phenotype, evidenced by the loss of α-smooth muscle actin expression upon encapsulation. In an effort to promote VIC-mediated ECM production, ascorbic acid (AA) was supplemented in the medium to investigate its effects on VIC function and phenotype. AA treatment enhanced VIC spreading and proliferation, and inhibited apoptosis. AA treatment also promoted VIC-mediated ECM remodelling by increasing MMP-2 activity and depositing collagens I and III. AA treatment did not significantly influence the expression of α-smooth muscle actin (myofibroblast activation marker) and alkaline phosphatase (osteogenic differentiation marker). No calcification or nodule formation was observed within the cell-laden hydrogels, with or without AA treatment. These results suggest the potential of this system and the beneficial effect of AA in heart valve tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

[Mechanism of tanshinone II A in inhibiting transformation of aortic valvular myofibroblast to osteoblast-like phenotype].

Aortic valve calcification (AVC) is a pathological process correlated with multiple disease causes and actively regulated by cardiac valve cells. In this study, porcine aortic valve myofibroblasts cultured in vitro were treated with 50 µg z L(-1) of pathological factor tumor necrosis factor α (TNF-α). Tanshinone II A (TSN) with the concentration of 50 mg x L(-1) and TNF-α were combined in incubating cells for 72 h (3 d) and 120 h (5 d). The Western blotting and Real-time PCR were adopted to detect the changes in smooth muscle α actin (α-SMA), bone morphogenetic protein 2 ( BMP2), alkaline phosphatase (ALP) in cells, and expressions of key effect proteins GSK-3ß and ß-catenin on Wnt/ß-catenin signal pathway. According to the findings, TNF-α can significantly increase the expression of myofibroblasts α-SMA and add the transformation activity to them, with nearly no expression of BMP2, ALP and mRNA in the control group and the TSN group but significant increase in their expressions in the TNF-α group (P < 0.01), which showed osteoblast-like phenotype. Moreover, TNF-α down-regulated the expression of up-streaming regulator GSK-3ß and mRNA expression (P < 0. 01) , notably increased the expression of key effect protein ß-catenin, but with no significant difference in mRNA with the control group and the TSN group. The result demonstrated that TSN showed a certain inhibitory effect on TNF-α's pathological impact (P < 0.05) in a time-dependent manner. Inflammatory factor TNF-α may promote the transformation of aortic valvular myofibroblasts to osteoblast-like phenotype by activating Wnt/ß-catenin signal pathway in aortic valvular myofibroblasts, so as to cause AVC. Tanshinone II A can have a preventive effect in AVC by activating GSK-3ß proteins and regulating signal transduction of Wnt/ß-catenin signal pathway.

Calcium and phosphorus concentrations in native and decellularized semilunar valve tissues.

BACKGROUND AND AIM OF THE STUDY: Native, allograft, xenograft and bioprosthetic semilunar valves are all susceptible to calcific degeneration. However, intrinsic differences in baseline calcium and phosphorus tissue concentrations within mammalian normal valve structural components (e.g., cusps, sinus, vessel wall) additionally subdivided by tripartite regions (e.g., right-, left- and non-coronary leaflets) have never been systematically measured and reported. It was originally hypothesized that variations in normative tissue concentrations of calcium and phosphorus may correspond to subsequent clinical patterns of acquired dystrophic calcification; decellularization was also expected to reduce the tissue concentrations of these elements. METHODS: Native semilunar valves were freshly harvested from 12 juvenile sheep. Half of the valves were decellularized (six aortic and six pulmonary), while the other valves were flash-frozen at -80 degrees C within minutes of euthanasia as native valves. Elemental calcium and phosphorus concentrations were measured in the great vessels, sinus walls and cusps using inductively coupled plasma optical emission spectrometry (ICP-OES), and analyzed with non-parametric statistical tests. RESULTS: Calcium concentrations (microg/mg tissue; median (range) were similar in aortic native cusps (0.37 (0.21)), sinus walls (0.37 (0.09)) and aorta (0.37 (0.08)) (p = 0.8298). Pulmonary calcium concentrations were similar in cusps, but 10-25% higher in the native sinus (p = 0.0018) and pulmonary artery (p < 0.0001) compared to analogous aortic structures. All cusps had higher phosphorus concentrations than their respective conduit tissues. No tripartite regional variations were observed. Decellularization did not reduce the calcium content of cusps, but removed 50-55% of vessel and sinus wall calcium. However, up to 85% of phosphorus was removed from all valve tissues (p < 0.001). CONCLUSION: There were no significant differences in normal tissue concentrations of calcium between aortic valve functional structures, and no semilunar tripartite regional differences in either semilunar valve complex. Thus, the distribution of baseline tissue calcium content of healthy young valves is not inherently predictive of selective or asymmetric anatomical patterns of valve degenerative calcification. Native semilunar cusps contain the highest phosphorus concentrations. Decellularization reduces all elemental concentrations except for cuspal calcium.

Characterization of porcine aortic valvular interstitial cell 'calcified' nodules.

Valve interstitial cells populate aortic valve cusps and have been implicated in aortic valve calcification. Here we investigate a common in vitro model for aortic valve calcification by characterizing nodule formation in porcine aortic valve interstitial cells (PAVICs) cultured in osteogenic (OST) medium supplemented with transforming growth factor beta 1 (TGF-ß1). Using a combination of materials science and biological techniques, we investigate the relevance of PAVICs nodules in modeling the mineralised material produced in calcified aortic valve disease. PAVICs were grown in OST medium supplemented with TGF-ß1 (OST+TGF-ß1) or basal (CTL) medium for up to 21 days. Murine calvarial osteoblasts (MOBs) were grown in OST medium for 28 days as a known mineralizing model for comparison. PAVICs grown in OST+TGF-ß1 produced nodular structures staining positive for calcium content; however, micro-Raman spectroscopy allowed live, noninvasive imaging that showed an absence of mineralized material, which was readily identified in nodules formed by MOBs and has been identified in human valves. Gene expression analysis, immunostaining, and transmission electron microscopy imaging revealed that PAVICs grown in OST+TGF-ß1 medium produced abundant extracellular matrix via the upregulation of the gene for Type I Collagen. PAVICs, nevertheless, did not appear to further transdifferentiate to osteoblasts. Our results demonstrate that 'calcified' nodules formed from PAVICs grown in OST+TGF-ß1 medium do not mineralize after 21 days in culture, but rather they express a myofibroblast-like phenotype and produce a collagen-rich extracellular matrix. This study clarifies further the role of PAVICs as a model of calcification of the human aortic valve.

Rapid 3D printing of anatomically accurate and mechanically heterogeneous aortic valve hydrogel scaffolds.

The aortic valve exhibits complex three-dimensional (3D) anatomy and heterogeneity essential for the long-term efficient biomechanical function. These are, however, challenging to mimic in de novo engineered living tissue valve strategies. We present a novel simultaneous 3D printing/photocrosslinking technique for rapidly engineering complex, heterogeneous aortic valve scaffolds. Native anatomic and axisymmetric aortic valve geometries (root wall and tri-leaflets) with 12-22 mm inner diameters (ID) were 3D printed with poly-ethylene glycol-diacrylate (PEG-DA) hydrogels (700 or 8000 MW) supplemented with alginate. 3D printing geometric accuracy was quantified and compared using Micro-CT. Porcine aortic valve interstitial cells (PAVIC) seeded scaffolds were cultured for up to 21 days. Results showed that blended PEG-DA scaffolds could achieve over tenfold range in elastic modulus (5.3±0.9 to 74.6±1.5 kPa). 3D printing times for valve conduits with mechanically contrasting hydrogels were optimized to 14 to 45 min, increasing linearly with conduit diameter. Larger printed valves had greater shape fidelity (93.3±2.6, 85.1±2.0 and 73.3±5.2% for 22, 17 and 12 mm ID porcine valves; 89.1±4.0, 84.1±5.6 and 66.6±5.2% for simplified valves). PAVIC seeded scaffolds maintained near 100% viability over 21 days. These results demonstrate that 3D hydrogel printing with controlled photocrosslinking can rapidly fabricate anatomical heterogeneous valve conduits that support cell engraftment.

Effects of dialdehyde starch on calcification of collagen matrix.

Dialdehyde starch (DAS), a polymeric aldehyde derived from naturally occurring polysaccharide, was used as an additive to the collagen (COL) matrix in an effort to improve its physical and biological properties. The effects of DAS on the thermal stability of COL were characterized by differential scanning calorimetry. The conformational changes in COL were characterized by resistance to protease degradation assay, residual amine analysis, and Fourier-transform infrared spectroscopy. To assess biocompatibility enhancement, the calcium content in porcine aortic valve interstitial cells (PAVICs) on exposure to cardiovascular stents coated with DAS-stabilized COL was examined using atomic absorption spectroscopy. Thermal stability of DAS-stabilized COL was affected by DAS in a concentration dependent manner, reaching maximum at the saturation concentration (DAS:COL = 1:120) and decreasing the thermal stability at the concentrations above saturation. In the long-term exposure condition (21 days), stents coated with DAS at the lowest concentration significantly reduced the calcification rate of PAVIC, when compared with bare stent (p = 0.001). DAS appears to be an efficient additive to the COL matrix in improving its physical and biological properties. Further optimization process is needed for the suitable crosslinking conditions of DAS, which subsequently enhances thermal strength and anticalcification activities of COL matrix.

Elevated cyclic stretch induces aortic valve calcification in a bone morphogenic protein-dependent manner.

Calcified aortic valve (AV) cusps have increased expression of bone morphogenic proteins (BMPs) and transforming growth factor-beta1 (TGF-beta1). Elevated stretch loading on the AV is known to increase expression of matrix remodeling enzymes and pro-inflammatory proteins. Little, however, is known about the mechanism by which elevated stretch might induce AV calcification. We investigated the hypothesis that elevated stretch may cause valve calcification via a BMP-dependent mechanism. Porcine AV cusps were cultured in a stretch bioreactor, at 10% (physiological) or 15% (pathological) stretch and 70 beats per minute for 3, 7, and 14 days, in osteogenic media supplemented with or without high phosphate (3.8 mmol/L), TGF-beta1 (1 ng/ml), as well as the BMP inhibitor noggin (1, 10, and 100 ng/ml). Fresh cusps served as controls. Alizarin red and von Kossa staining demonstrated that 15% stretch elicited a stronger calcification response compared with 10% stretch in a fully osteogenic medium containing high phosphate and TGF-beta1. BMP-2, -4, and Runx2 expression was observed after 3 days on the fibrosa surface of the valve cusp and was stretch magnitude-dependent. Cellular apoptosis was highest at 15% stretch. Tissue calcium content and alkaline phosphatase activity were similarly stretch-dependent and were significantly reduced by noggin in a dose dependent manner. These results underline the potential role of BMPs in valve calcification due to altered stretch.

A novel role of extracellular nucleotides in valve calcification: a potential target for atorvastatin.

BACKGROUND: Calcific aortic valve disease is a common condition and is associated with inflammatory changes and expression of osteoblast-like cell phenotypes, but the cellular mechanisms are unclear. Recent studies identified extracellular ATP and P2Y receptor cascade as important regulators of bone remodeling, whereas its breakdown product, adenosine, is known to have anti-inflammatory properties. We hypothesize that extracellular ATP and adenosine have important roles in regulating osteoblast differentiation in human valve interstitial cells, and that this can be a potential target for therapy. Method and Results- Primary cultures of human valve interstitial cells (ICs) treated for 21 days with osteogenic media, ATP, and ATP-gamma-S (a stable agonist of the P2Y receptor) revealed a significant increase in alkaline phosphatase (ALP) (an osteoblast marker) activity and expression as measured using spectrophotometric assay and immunocytochemistry staining. Valve ICs treated with adenosine alone did not cause an increase in ALP activity; however, adenosine treatment decreased the ALP activity and expression induced by osteogenic media after 21 days of incubation. In addition, atorvastatin inhibited the activity of ALP induced by ATP in human valve ICs, and enzyme studies revealed that atorvastatin upregulated the breakdown of extracellular ATP into adenosine in human valve ICs after 24-hour treatment. CONCLUSIONS: These findings identify a novel role for extracellular nucleotides in inducing osteoblast differentiation in human valve ICs in vitro and provide a potential therapeutic target for preventing the disease progression.

Calcification of human valve interstitial cells is dependent on alkaline phosphatase activity.

BACKGROUND AND AIM OF THE STUDY: The calcification of heart valves is associated with valve degeneration and failure, but the mechanisms involved are poorly understood. The presence of lamellar bone has been demonstrated in calcified aortic valves. Since osseous calcification is closely associated with alkaline phosphatase (ALP) activity, it was hypothesized that ALP activity might be implicated in the calcification of isolated leaflet interstitial cells (ICs). METHODS: Human valve leaflet ICs were isolated from transplant-explanted hearts at the time of transplantation (n = 12). RESULTS: Isolated leaflet ICs expressed the fibroblast-specific antigen (100% of cells) and smooth muscle (SM) alpha-actin (70-80% of cells), but osteoblastic markers were not expressed. Cultured ICs did not calcify spontaneously, however when the growth medium was supplemented with beta-glycerophosphate (an organic phosphate) it induced the formation of calcified nodules that expressed osteonectin and ALP, but not SM alpha-actin. Beta-glycerophosphate-induced calcification of ICs showed a time-dependent effect on the calcium content of treated cells over a 14-day period. ALP activity was considerably increased in beta-glycerophosphate-treated ICs, and this correlated with the calcium content (r = 0.5: p = 0.01). Levamisol (an ALP inhibitor) inhibited the beta-glycerophosphate-induced calcification process, as well as the expression of osteoblastic differentiation markers. CONCLUSION: Isolated and cultured leaflet ICs did not calcify spontaneously, though organic phosphate induced the formation of calcified nodules that expressed osteoblastic markers. The calcification of isolated ICs was seen to be dependent on ALP activity.

Evidence for the presence of endothelin ETA receptors in endothelial cells in situ on the aortic side of porcine aortic valve.

1. In the present study, we determined whether ETA receptors are present on endothelial cells in situ, by use of front-surface fluorometry of fura-2-loaded porcine aortic valvular strips and reverse transcription polymerase chain reaction (RT-PCR). 2. Although endothelin-1 (ET-1) and endothelin-3 (ET-3) induced maximum elevations of cytosolic Ca2+ concentrations ([Ca2+]i) at 10(-7) M, the peak elevations of [Ca2+]i induced by ET-1 were much greater than those induced by ET-3. 3. The application of ET-1 after ET-3 induced an additional increase in [Ca2+]i, while the application of ET-3 after ET-1 had no effect. A selective ETA receptor antagonist, BQ-123, partially inhibited the ET-1-induced Ca2+ transient but had no effect on ET-3-induced Ca2+ transients. These experiments indicated the presence of functioning ETA receptors in addition to ETB receptors in endothelial cells in situ. 4. The sequence of pig lung ETA receptor complimentary DNA (cDNA) was determined by PCR. RT-PCR, using specific primers for pig ETA receptor sequence and total RNA from endothelial cells on the aortic side of the aortic valve, gave the expected size of band. This PCR product was sequenced and was found to be identical to the sequence of the pig lung ETA receptor. 5. The partial sequence of the pig lung ETB receptor was also determined. RT-PCR for the pig ETB receptor revealed that endothelial cells of the aortic valve express ETB receptor messenger RNA (mRNA). 6. We confirmed that functioning ETA receptors and expression of ETA receptor mRNA exist in the endothelial cells on the aortic side of porcine aortic valves.