Background—: Progerin is a mutant form of lamin A responsible for Hutchinson-Gilford progeria syndrome (HGPS), a premature aging disorder characterized by excessive atherosclerosis and vascular calcification that leads to premature death, predominantly of myocardial infarction or stroke. The goal of this study was to investigate mechanisms that cause excessive vascular calcification in HGPS. Methods and Results—: We performed expression and functional studies in wild-type mice and knock-in Lmna ^G609G/+ mice expressing progerin, which mimic the main clinical manifestations of HGPS. Lmna ^G609G/+ mice showed excessive aortic calcification, and primary aortic vascular smooth muscle cells from these progeroid animals had an impaired capacity to inhibit vascular calcification. This defect in progerin-expressing vascular smooth muscle cells is associated with increased expression and activity of tissue-nonspecific alkaline phosphatase and mitochondrial dysfunction, which leads to reduced ATP synthesis. Accordingly, Lmna ^G609G/+ vascular smooth muscle cells are defective for the production and extracellular accumulation of pyrophosphate, a major inhibitor of vascular calcification. We also found increased alkaline phosphatase activity and reduced ATP and pyrophosphate levels in plasma of Lmna ^G609G/+ mice without changes in phosphorus and calcium. Treatment with pyrophosphate inhibited vascular calcification in progeroid mice. Conclusions—: Excessive vascular calcification in Lmna ^G609G mice is caused by reduced extracellular accumulation of pyrophosphate that results from increased tissue-nonspecific alkaline phosphatase activity and diminished ATP availability caused by mitochondrial dysfunction in vascular smooth muscle cells. Excessive calcification is ameliorated on pyrophosphate treatment. These findings reveal a previously undefined pathogenic process in HGPS that may also contribute to vascular calcification in normal aging, because progerin progressively accumulates in the vascular tissue of individuals without HGPS.

The principal mediators of renal phosphate (P_i) reabsorption are the SLC34 family proteins NaPi-IIa and NaPi-IIc, localized to the proximal tubule (PT) apical membrane. Their abundance is regulated by circulatory factors and dietary P_i. Although their physiological importance has been confirmed in knockout animal studies, significant P_ireabsorptive capacity remains, which suggests the involvement of other secondary-active P_itransporters along the nephron. Here we show that a member of the SLC20 gene family (PiT-2) is localized to the brush-border membrane (BBM) of the PT epithelia and that its abundance, confirmed by Western blot and immunohistochemistry of rat kidney slices, is regulated by dietary P_i. In rats treated chronically on a high-P_i(1.2%) diet, there was a marked decrease in the apparent abundance of PiT-2 protein in kidney slices compared with those from rats kept on a chronic low-P_i(0.1%) diet. In Western blots of BBM from rats that were switched from a chronic low- to high-P_idiet, NaPi-IIa showed rapid downregulation after 2 h; PiT-2 was also significantly downregulated at 24 h and NaPi-IIc after 48 h. For the converse dietary regime, NaPi-IIa showed adaptation within 8 h, whereas PiT-2 and NaPi-IIc showed a slower adaptive trend. Our findings suggest that PiT-2, until now considered as a ubiquitously expressed P_ihousekeeping transporter, is a novel mediator of P_ireabsorption in the PT under conditions of acute P_ideprivation, but with a different adaptive time course from NaPi-IIa and NaPi-IIc.

In this work we are studying whether calcium phosphate deposition (CPD) during vascular calcification is a passive or a cell-mediated mechanism. Passive CPD was studied in fixed vascular smooth muscle cells (VSMC), which calcify faster than live cells in the presence of 1.8 mM Ca²⁺ and 2 mM P_i. CPD seems to be a cell-independent process that depends on the concentration of calcium, phosphate, and hydroxyl ions, but not on Ca × P_i concentration products, given that deposition is obtained with 2 × 2 and 4 × 1 Ca × P_i mM² but not with 2 × 1 or 1 × 4 Ca × P_i mM². Incubation with 4 mM P_i without CPD (i.e., plus 1 mM Ca) does not induce osteogene expression. Increased expression of bone markers such as Bmp2 and Cbfa1 is only observed concomitantly with CPD. Hydroxyapatite is the only crystalline phase in both lysed and live cells. Lysed cell deposits are highly crystalline, whereas live cell deposits still contain large amounts of amorphous calcium. High-resolution transmission electron microscopy revealed a nanostructure of rounded crystallites of 5–10 nm oriented at random in lysed cells, which is compatible with spontaneous precipitation. The nanostructure in live cells consisted of long fiber crystals, 10-nm thick, embedded in an amorphous matrix. This structure indicates an active role of cells in the process of hydroxyapatite crystallization. In conclusion, our data suggest that CPD is a passive phenomenon, which triggers the osteogenic changes that are involved in the formation of a well organized, calcified crystalline structure.

Background: Progerin, an aberrant protein that accumulates with age, causes the rare genetic disease Hutchinson-Gilford progeria syndrome (HGPS). Patients who have HGPS exhibit ubiquitous progerin expression, accelerated aging and atherosclerosis, and die in their early teens, mainly of myocardial infarction or stroke. The mechanisms underlying progerin-induced atherosclerosis remain unexplored, in part, because of the lack of appropriate animal models. Methods: We generated an atherosclerosis-prone model of HGPS by crossing apolipoprotein E–deficient ( Apoe ^–/– ) mice with Lmna ^G609G/G609G mice ubiquitously expressing progerin. To induce progerin expression specifically in macrophages or vascular smooth muscle cells (VSMCs), we crossed Apoe ^–/– Lmna ^LCS/LCS mice with LysMCre and SM22αCre mice, respectively. Progerin expression was evaluated by polymerase chain reaction and immunofluorescence. Cardiovascular alterations were determined by immunofluorescence and histology in male mice fed normal chow or a high-fat diet. In vivo low-density lipoprotein retention was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein. Cardiac electric defects were evaluated by electrocardiography. Results: Apoe ^–/– Lmna ^G609G/G609G mice with ubiquitous progerin expression exhibited a premature aging phenotype that included failure to thrive and shortened survival. In addition, high-fat diet–fed Apoe ^–/– Lmna ^G609G/G609G mice developed a severe vascular pathology, including medial VSMC loss and lipid retention, adventitial fibrosis, and accelerated atherosclerosis, thus resembling most aspects of cardiovascular disease observed in patients with HGPS. The same vascular alterations were also observed in Apoe ^–/– Lmna ^LCS/LCS SM22αCre mice expressing progerin specifically in VSMCs, but not in Apoe ^–/– Lmna ^LCS/LCS LysMCre mice with macrophage-specific progerin expression. Moreover, Apoe ^–/– Lmna ^LCS/LCS SM22αCre mice had a shortened lifespan despite the lack of any overt aging phenotype. Aortas of ubiquitously and VSMC-specific progerin-expressing mice exhibited increased retention of fluorescently labeled human low-density lipoprotein, and atheromata in both models showed vulnerable plaque features. Immunohistopathological examination indicated that Apoe ^–/– Lmna ^LCS/LCS SM22αCre mice, unlike Apoe ^–/– Lmna ^G609G/G609G mice, die of atherosclerosis-related causes. Conclusions: We have generated the first mouse model of progerin-induced atherosclerosis acceleration, and demonstrate that restricting progerin expression to VSMCs is sufficient to accelerate atherosclerosis, trigger plaque vulnerability, and reduce lifespan. Our results identify progerin-induced VSMC death as a major factor triggering atherosclerosis and premature death in HGPS.

Extracellular inorganic pyrophosphate (ePP_i) is an important endogenous inhibitor of vascular calcification, but it is not known whether systemic or local vascular PP_imetabolism controls calcification. To determine the role of ePP_iin vascular smooth muscle, we identified the pathways responsible for ePP_iproduction and hydrolysis in rat and mouse aortas and manipulated them to demonstrate their role in the calcification of isolated aortas in culture. Rat and mouse aortas contained mRNA for ectonucleotide pyrophosphatase/phosphodiesterases (NPP1–3), the putative PP_itransporter ANK, and tissue-nonspecific alkaline phosphatase (TNAP). Synthesis of PP_ifrom ATP in aortas was blocked by β,γ-methylene-ATP, an inhibitor of NPPs. Aortas from mice lacking NPP1 ( Enpp1^−/−) did not synthesize PP_ifrom ATP and exhibited increased calcification in culture. Although ANK-mediated transport of PP_icould not be demonstrated in aortas, aortas from mutant ( ank/ank) mice calcified more in culture than did aortas from normal (ANK/ANK) mice. Hydrolysis of PP_iwas reduced 25% by β,γ-methylene-ATP and 50% by inhibition of TNAP. Hydrolysis of PP_iwas increased in cells overexpressing TNAP or NPP3 but not NPP1 and was not reduced in Enpp1^−/−aortas. Overexpression of TNAP increased calcification of cultured aortas. The results show that smooth muscle NPP1 and TNAP control vascular calcification through effects on synthesis and hydrolysis of ePP_i, indicating an important inhibitory role of locally produced PP_i. Smooth muscle ANK also affects calcification, but this may not be mediated through transport of PP_i. NPP3 is identified as an additional pyrophosphatase that could influence vascular calcification.

Cardiovascular complications due to accelerated arterial stiffening and atherosclerosis are the leading cause of morbimortality in Western society. Both pathologies are frequently associated with vascular calcification. Pathologic calcification of cardiovascular structures, or vascular calcification, is associated with several diseases (for example, genetic diseases, diabetes, and chronic kidney disease) and is a common consequence of aging. Calcium phosphate deposition, mainly in the form of hydroxyapatite, is the hallmark of vascular calcification and can occur in the medial layer of arteries (medial calcification), in the atheroma plaque (intimal calcification), and cardiac valves (heart valve calcification). Although various mechanisms have been proposed for the pathogenesis of vascular calcification, our understanding of the pathogenesis of calcification is far from complete. However, in recent years, some risk factors have been identified, including high serum phosphorus concentration (hyperphosphatemia) and defective synthesis of pyrophosphate (pyrophosphate deficiency). The balance between phosphate and pyrophosphate, strictly controlled by several genes, plays a key role in vascular calcification. This review summarizes the current knowledge concerning phosphate and pyrophosphate homeostasis, focusing on the role of extracellular pyrophosphate metabolism in aortic smooth muscle cells and macrophages.