New calcification model for intact murine aortic valves.

Calcific aortic valve disease (CAVD) is a common progressive disease of the aortic valves, for which no medical treatment exists and surgery represents currently the only therapeutic solution. The development of novel pharmacological treatments for CAVD has been hampered by the lack of suitable test-systems, which require the preservation of the complex valve structure in a mechanically and biochemical controllable system. Therefore, we aimed at establishing a model which allows the study of calcification in intact mouse aortic valves by using the Miniature Tissue Culture System (MTCS), an ex vivo flow model for whole mouse hearts. Aortic valves of wild-type mice were cultured in the MTCS and exposed to osteogenic medium (OSM, containing ascorbic acid, ß-glycerophosphate and dexamethasone) or inorganic phosphates (PI). Osteogenic calcification occurred in the aortic valve leaflets that were cultured ex vivo in the presence of PI, but not of OSM. In vitro cultured mouse and human valvular interstitial cells calcified in both OSM and PI conditions, revealing in vitro-ex vivo differences. Furthermore, endochondral differentiation occurred in the aortic root of ex vivo cultured mouse hearts near the hinge of the aortic valve in both PI and OSM conditions. Dexamethasone was found to induce endochondral differentiation in the aortic root, but to inhibit calcification and the expression of osteogenic markers in the aortic leaflet, partly explaining the absence of calcification in the aortic valve cultured with OSM. The osteogenic calcifications in the aortic leaflet and the endochondral differentiation in the aortic root resemble calcifications found in human CAVD. In conclusion, we have established an ex vivo calcification model for intact wild-type murine aortic valves in which the initiation and progression of aortic valve calcification can be studied. The in vitro-ex vivo differences found in our studies underline the importance of ex vivo models to facilitate pre-clinical translational studies.

Superimposed Tissue Formation in Human Aortic Valve Disease: Differences between Regurgitant and Stenotic Valves.

The formation of superimposed tissue (SIT), a layer on top of the original valve leaflet, has been described in patients with mitral regurgitation as a major contributor to valve thickening and possibly as a result of increased mechanical stresses. However, little is known whether SIT formation also occurs in aortic valve disease. We therefore performed histological analyses to assess SIT formation in aortic valve leaflets (n = 31) from patients with aortic stenosis (n = 17) or aortic regurgitation due to aortic dilatation (n = 14). SIT was observed in both stenotic and regurgitant aortic valves, both on the ventricular and aortic sides, but with significant differences in distribution and composition. Regurgitant aortic valves showed more SIT formation in the free edge, leading to a thicker leaflet at that level, while stenotic aortic valves showed relatively more SIT formation on the aortic side of the body part of the leaflet. SIT appeared to be a highly active area, as determined by large populations of myofibroblasts, with varied extracellular matrix composition (higher collagen content in stenotic valves). Further, the identification of the SIT revealed the presence of foldings of the free edge in the diseased aortic valves. Insights into SIT regulation may further help in understanding the pathophysiology of aortic valve disease and potentially lead to the development of new therapeutic treatments.

Differential effect of morphine and morphine-6-glucuronide on the control of breathing in the anesthetized cat.

BACKGROUND: Morphine's metabolite, morphine-6-glucuronide (M6G), activates the mu-opioid receptor. Previous data suggest that M6G activates a unique M6G receptor that is selectively antagonized by 3-methoxynaltrexome (3mNTX). The authors compared the effects of M6G and morphine on breathing in the anesthetized cat and assessed whether 3mNTX reversal was selective for M6G. METHODS: Step changes in end-tidal carbon dioxide concentration were applied in cats anesthetized with alpha-chloralose-urethane. In study 1, the effect of the 0.15 mg/kg morphine followed by 0.2 mg/kg 3mNTX and next 0.8 mg/kg M6G was assessed in six cats. In study 2, the effect of 0.8 mg/kg M6G followed by 0.2 mg/kg 3mNTX and 0.15 mg/kg morphine was tested in another six cats. The ventilatory carbon dioxide responses were analyzed with a two-compartment model of the ventilatory controller, which consists of a fast peripheral and a slow central component. RESULTS: Both opioids shifted the ventilatory carbon dioxide responses to higher end-tidal carbon dioxide levels. Morphine had a preferential depressant effect within the central chemoreflex loop. In contrast, M6G had a preferential depressant effect within the peripheral chemoreflex loop. Irrespective of the opioid, 3mNTX caused full reversal of and prevented respiratory depression. CONCLUSIONS: In anesthetized cats, the mu-opioids morphine and M6G induce respiratory depression at different sites within the ventilatory control system. Because 3mNTX caused full reversal of the respiratory depressant effects of both opioids, it is unlikely that a 3mNTX-sensitive unique M6G receptor is the cause of the differential respiratory behavior of morphine and M6G.

The carbonic anhydrase inhibitors methazolamide and acetazolamide have different effects on the hypoxic ventilatory response in the anaesthetized cat.

We compared the effects of the carbonic anhydrase inhibitors methazolamide and acetazolamide (3 mg kg(-1), i.v.) on the steady-state hypoxic ventilatory response in 10 anaesthetized cats. In five additional animals, we studied the effect of 3 and 33 mg kg(-1) methazolamide. The steady-state hypoxic ventilatory response was described by the exponential function: *Vi= G exp(-D P(O2)) + A where *Vi is the inspired ventilation, G is hypoxic sensitivity, D is the shape factor and A is hyperoxic ventilation. In the first group of 10 animals, methazolamide did not change parameters G and D, while A increased from 0.86 +/- 0.33 to 1.30 +/- 0.40 l min(-1) (mean +/- s.d., P = 0.003). However, the subsequent administration of acetazolamide reduced G by 44% (control, 1.93 +/- 1.32; acetazolamide, 1.09 +/- 0.92 l min(-1), P = 0.003), while A did not show a further change. Acetazolamide tended to reduce D (control, 0.20 +/- 0.07; acetazolamide, 0.14 +/- 0.06 kPa(-1), P = 0.023). In the second group of five animals, neither low- nor high-dose methazolamide changed parameters G, D and A. The observation that even high-dose methazolamide, causing full inhibition of carbonic anhydrase in all body tissues, did not reduce the hypoxic ventilatory response is reminiscent of previous findings by others showing no change in magnitude of the hypoxic response of the in vitro carotid body by this agent. This suggests that normal carbonic anhydrase activity is not necessary for a normal hypoxic ventilatory response to occur. The mechanism by which acetazolamide reduces the hypoxic ventilatory response needs further study.

In vivo temperature heterogeneity is associated with plaque regions of increased MMP-9 activity.

AIMS: Plaque rupture has been associated with a high matrix metalloproteinase (MMP) activity. Recently, regional temperature variations have been observed in atherosclerotic plaques in vivo and ascribed to the presence of macrophages. As macrophages are a major source of MMPs, we examined whether regional temperature changes are related to local MMP activity and macrophage accumulation. METHODS AND RESULTS: Plaques were experimentally induced in rabbit (n=11) aortas, and at the day of sacrifice, a pull-back was performed with a thermography catheter. Hot (n=10), cold (n=10), and reference (n=11) regions were dissected and analysed for smooth muscle cell (SMC), lipids (L), collagen (COL), and macrophage (MPhi) cell densities (%); a vulnerability index (VI) was calculated as VI=MPhi+L/(SMC+COL). In addition, accumulation and activity of MMP-2 and MMP-9 were determined with zymography. Ten hot regions were identified with an average temperature of 0.40+/-0.03 degrees C (P<0.05 vs. reference) and 10 cold regions with 0.07+/-0.03 degrees C (P<0.05 vs. hot). In the hot regions, a higher macrophage density (173%), less SMC density (77%), and a higher VI (100%) were identified. In addition, MMP-9 (673%) activity was increased. A detailed regression analysis revealed that MMP-9 predicted hot regions better than macrophage accumulation alone. CONCLUSION: In vivo temperature measurements enable to detect plaques that contain more macrophages, less SMCs, and a higher MMP-9 activity.

Intravascular thermography: Immediate functional and morphological vascular findings.

AIMS: To investigate safety, feasibility, and injurious effect on endothelial cells of a thermography catheter as well as effect of flow on measured temperature in non-obstructive arteries. METHODS AND RESULTS: Safety and feasibility were tested in both rabbit aortas and pig coronary arteries. Evaluation of endothelial damage by the catheter (acute, 7 and 14 days) was performed in pig coronaries using Evans Blue, scanning electron microscopy (SEM) and Factor-VIII antibody and compared with normal arteries and arteries that underwent intravascular ultrasound (IVUS). The effect of flow on temperature heterogeneity was analysed both in vitro and in vivo conditions. All procedures were successful without any adverse events; intra- and inter-operator variability was low. Intracoronary use of the catheter was associated with acute but reversible de-endothelialization, paralleling the findings associated with IVUS use. Changes in flow velocities under physiologic flow conditions did not significantly influence the temperature differences measured both in vitro and in vivo; temperature heterogeneity was more pronounced in absence of flow. CONCLUSIONS: Intracoronary thermography using a dedicated catheter is safe and feasible with a similar degree of de-endothelialization as IVUS. Temperature heterogeneity remained unchanged under normal physiologic flow conditions allowing clinical use of thermography.

The role of shear stress in atherosclerosis: action through gene expression and inflammation?

Atherosclerotic lesions preferentially localize near side branches or curved vessels. During the last few decades, research has been shown that low or low and oscillating shear stress is associated with plaque location. Despite ample evidence, the precise mechanism is unknown. This is mainly because of a lack of appropriate animal models. We describe two novel methods to study the hypothesis that shear stress acts through endothelial gene expression or shear stress acts through localizing of inflammation. Both literature evidence and own findings support a role for both mechanisms in atherosclerosis.