Simon Moreau

Sequential Activation of Matrix Metalloproteinase 9 and Transforming Growth Factor β in Arterial Elastocalcinosis

Objective—Isolated systolic hypertension is associated with increased elastase activity, vascular calcification, and vascular stiffness. We sought to determine the importance of elastase activity and matrix degradation in the development of elastocalcinosis. Methods and Results—Elastocalcinosis was induced in vivo and ex vivo using warfarin. Hemodynamic parameters, calcium deposition, elastin degradation, transforming growth factor (TGF)-&bgr; signaling, and elastase activity were evaluated at different time points in the in vivo model. Metalloproteinases, serine proteases, and cysteine proteases were blocked to measure their relative implication in elastin degradation. Gradual elastocalcinosis was obtained, and paralleled the elastin degradation pattern. Matrix metalloproteinase (MMP)-9 activity was increased at 5 days of warfarin treatment, whereas TGF-&bgr; signaling was increased at 7 days. Calcification was significantly elevated after 21 days. Blocking metalloproteinases activation with doxycycline and TGF-&bgr; signaling with SB-431542 were able to prevent calcification. Conclusions—Early MMP-9 activation precedes the increase of TGF-&bgr; signaling, and overt vascular elastocalcinosis and stiffness. Modulation of matrix degradation could represent a novel therapeutic avenue to prevent the gradual age-related stiffening of large arteries, leading to isolated systolic hypertension.

Reduction of Advanced-Glycation End Products Levels and Inhibition of RAGE Signaling Decreases Rat Vascular Calcification Induced by Diabetes

Advanced-glycation end products (AGEs) were recently implicated in vascular calcification, through a process mediated by RAGE (receptor for AGEs). Although a correlation between AGEs levels and vascular calcification was established, there is no evidence that reducing in vivo AGEs deposition or inhibiting AGEs-RAGE signaling pathways can decrease medial calcification. We evaluated the impact of inhibiting AGEs formation by pyridoxamine or elimination of AGEs by alagebrium on diabetic medial calcification. We also evaluated if the inhibition of AGEs-RAGE signaling pathways can prevent calcification. Rats were fed a high fat diet during 2 months before receiving a low dose of streptozotocin. Then, calcification was induced with warfarin. Pyridoxamine was administered at the beginning of warfarin treatment while alagebrium was administered 3 weeks after the beginning of warfarin treatment. Results demonstrate that AGEs inhibitors prevent the time-dependent accumulation of AGEs in femoral arteries of diabetic rats. This effect was accompanied by a reduced diabetes-accelerated calcification. Ex vivo experiments showed that N-methylpyridinium, an agonist of RAGE, induced calcification of diabetic femoral arteries, a process inhibited by antioxidants and different inhibitors of signaling pathways associated to RAGE activation. The physiological importance of oxidative stress was demonstrated by the reduction of femoral artery calcification in diabetic rats treated with apocynin, an inhibitor of reactive oxygen species production. We demonstrated that AGE inhibitors prevent or limit medial calcification. We also showed that diabetes-accelerated calcification is prevented by antioxidants. Thus, inhibiting the association of AGE-RAGE or the downstream signaling reduced medial calcification in diabetes.

Distinct effects of amlodipine treatment on vascular elastocalcinosis and stiffness in a rat model of isolated systolic hypertension

Objective Medial elastocalcinosis (MEC) contributes to the development of large artery stiffness and isolated systolic hypertension. Since endothelin receptor antagonists can prevent and regress elastocalcinosis, our aim was to determine whether amlodipine, a calcium channel blocker that inhibits endothelin signaling, could likewise influence MEC, or reduce pressure mainly through its vasorelaxing properties. Methods Control male Wistar rats were compared with rats receiving warfarin (20 mg/kg per day) with vitamin K1 (15 mg/kg per day) alone (WVK) or in association with amlodipine (15 mg/kg per day) for 4 weeks or during the last week or last 4 weeks of an 8-week WVK treatment (two regression groups). Results Inactivation of matrix Gla protein by WVK for 4 or 8 weeks increased the calcium content 10-fold in the aorta, inducing a significant elevation of pulse wave velocity and pulse pressure by selective augmentation of systolic blood pressure. Amlodipine prevented aortic MEC, pulse wave velocity and pulse pressure elevation, but reversed only MEC and pulse pressure when administered for 4 weeks. One week of amlodipine administered after 7 weeks of WVK partially decreased pulse pressure without modifying aortic MEC. Amlodipine did not reduce the fibrosis associated with calcified areas in the WVK model during the regression protocols. Conclusion The clinical efficacy of amlodipine in improving hemodynamic variables and reducing cardiovascular events in isolated systolic hypertension could be explained by its beneficial effect on vascular calcification. Amlodipines lack of effect on pulse wave velocity and collagen deposition, however, suggests that it may reduce pulse pressure by means other than improving arterial stiffness.

Fluorescent probes of tissue transglutaminase reveal its association with arterial stiffening.

Tissue transglutaminase (TG2) catalyzes the crosslinking of proteins. TG2 has been implicated in fibrosis and vascular calcification, both of which lead to a common feature of aging known as arterial stiffness. In order to probe the role of TG2 in arterial rigidification, we have prepared a fluorescent irreversible inhibitor as a probe for TG2 activity (RhodB-PGG-K(Acr)-LPF-OH). This probe was synthesized on solid support, characterized kinetically (k(inact) = 0.68 min⁻¹, K(I) = 79 μM), and then used to stain the aorta from rats used as a model of isolated systolic hypertension (ISH). Interestingly, TG2 activity was thus shown to increase over 4 weeks of the hypertension model, corresponding with the previously observed increase in arterial stiffness. These results clearly suggest an association between TG2 and the phenomenon of arterial rigidification.

Arterial Stiffness As A Therapeutic Target For Isolated Systolic Hypertension: Focus on Vascular Calcification and Fibrosis

Isolated systolic hypertension is the most common form of essential hypertension in patients over 65 years old and is not well controlled by current antihypertensive therapies. Current antihypertensive pharmacology is focused on reducing peripheral resistance and ventricular ejection. However, the increase of systolic blood pressure is mainly a consequence of large artery stiffening. This pathological process seems to be the result of medial arterial calcification (or elastocalcinosis), elastin degradation, extracellular matrix fibrosis and endothelial dysfunction. As a unifying hypothesis, we propose that initial extracellular calcification could promote extracellular matrix-cellular interactions by involving metalloproteinase matrix degradation, leading to the liberation of embedded transforming growth factor-β. This growth factor could promote a cascade of events involving vascular smooth muscle cells that adopt an osteogenic phenotype and express a different set of proteins, such as endothelin, that appear to play a central role in medial calcification and fibrosis. This review highlights the evidence supporting the hypothesis. It also presents the effects of current drugs on calcification and/or fibrosis in experimental model of isolated systolic hypertension to illustrate where we stand in our efforts to modify the process of arterial stiffening.