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)-␤ 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-␤ signaling was increased at 7 days. Calcification was significantly elevated after 21 days. Blocking metalloproteinases activation with doxycycline and TGF-␤ signaling with SB-431542 were able to prevent calcification. Conclusions-Early MMP-9 activation precedes the increase of TGF-␤ 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. Elastocalcinosis is characterized by a deposition of hydroxyapatite on the elastic lamellae of arteries. It occurs independently of atherosclerosis. 2 Until recently, it was considered as a passive process, taking place with time. However, several studies demonstrated that vascular calcification is an active phenomenon controlled by serum and matrix proteins, such as matrix Gla protein. 3 Moreover, it involves phenotypic changes of vascular smooth muscle cells with the expression of bone-related proteins. 4 Aging is associated with an increased collagen/elastin ratio explained in part by an enhanced degradation of elastin. 5,6 Indeed, elastase activity, mainly endopeptidases, including cysteine proteases, serine proteases, and metalloproteinases, is increased with age in human aortas. 7 In the context of aging and vascular calcification, metalloproteinases, especially matrix metalloproteinases (MMPs), have been thoroughly studied. Investigations on the genetic mutations of MMPs demonstrated its contribution to age-related large artery stiffening. 8,9 Furthermore, enhanced MMP-9 and MMP-2 levels and serum elastase activity were observed in patients with ISH and correlated independently with PWV. 10 In 2000, Vyavahare et al showed for the first time that elastin calcification was blunted by a site specific delivery of MMP inhibitor. 11 Moreover, Qin et al demonstrated, in an animal model, that matrix metalloproteinase inhibition with doxycycline and GM6001 decreased hydroxyapatite accumulation in the aorta. 12 These results suggest that MMPs and elastin degradation are involved in MEC.Elastin degradation induces the release of soluble elastin pep...

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.

BIM-44002, a pure competitive antagonist of parathyroid hormone (PTH), has a high affinity for the PTH/PTHrP receptor in vitro, and can completely inhibit the actions of a PTH agonist in rats in vivo. Toxicology studies in rats and dogs showed BIM-44002 to be devoid of any adverse effects. Therefore we undertook an investigation to evaluate the potential utility of BIM-44002 in lowering elevated serum calcium in three patients with primary hyperparathyroidism. BIM-44002 was administered by continuous intravenous infusion at dosages of 100 microg/hour (370 nmol/hour) for 12 hours, followed by 200 microg/hour for 12 hours, followed by 400 microg/hour for 12 hours. Vital signs and serum ionized and total calcium were monitored hourly and for 3 hours after cessation of the infusion. Blood for PTH determinations was obtained at the same time points. Serum calcium and PTH did not change during and after the infusion of the antagonist. No subject experienced any adverse reactions to the infusion of the antagonist. We conclude that although the PTH antagonist BIM-44002 was effective both in vitro and in vivo in animals, and it was safe in humans, it was not able to lower serum calcium in patients with hyperparathyroidism. Possible reasons for lack of clinical efficacy are discussed.

We have established a model of accelerated elastocalcinosis in diabetes related to its duration and localized in distal arteries. The modification of local protein expression is also in accordance with clinical data, suggesting that this model could be useful to investigate mechanisms related to this important clinical macrovascular complication of diabetes.

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.