Tineke M. De Schutter

Chondrocyte rather than osteoblast conversion of vascular cells underlies medial calcification in uremic rats

Objective—To investigate cell biological changes in calcified aortas of rats that experienced chronic renal failure. Methods and Results—Vascular smooth muscle cells have the potential to transdifferentiate to either chondrocytes or osteoblasts, depending on the molecular pathways that are stimulated. Uremia-related medial calcification was induced by feeding rats an adenine low-protein diet for 4 weeks. Aortic calcification was evaluated biochemically and histochemically and with in vivo micro–computed tomographic scanning. Immunohistochemistry and RT-PCR were applied to analyze the time-dependent aortic expression of molecules involved in the segregation between the chondrocyte versus osteoblast differentiation pathway. After 4 weeks, 85% of the uremic rats had developed distinct aortic medial calcification, which increased to severely calcified lesions during further follow-up. The calcification process was accompanied by a significant time-dependent increase in the expression of the chondrocyte-specific markers sex determining region Y-box 9 (sox9), collagen II, and aggrecan and a nonsignificant trend toward enhanced core binding factor alpha 1 (cbfa1), and collagen I. The expression of the osteoblast marker osterix and both lipoprotein receptor–related protein 6 and &bgr;-catenin, molecules of the wingless-type MMTV integration site family member (Wnt)/&bgr;-catenin pathway induced during osteoblast differentiation, was suppressed. Conclusion—In the aorta of uremic rats, medial smooth muscle cells acquire a chondrocyte rather than osteoblast phenotype during the calcification process.

Effect of a magnesium-based phosphate binder on medial calcification in a rat model of uremia

Calcium-based phosphate binders are used to control hyperphosphatemia; however, they promote hypercalcemia and may accelerate aortic calcification. Here we compared the effect of a phosphate binder containing calcium acetate and magnesium carbonate (CaMg) to that of sevelamer carbonate on the development of medial calcification in rats with chronic renal failure induced by an adenine diet for 4 weeks. After 1 week, rats with chronic renal failure were treated with vehicle, 375 or 750 mg/kg CaMg, or 750 mg/kg sevelamer by daily gavage for 5 weeks. Renal function was significantly impaired in all groups. Vehicle-treated rats with chronic renal failure developed severe hyperphosphatemia, but this was controlled in treated groups, particularly by CaMg. Neither CaMg nor sevelamer increased serum calcium ion levels. Induction of chronic renal failure significantly increased serum PTH, dose-dependently prevented by CaMg but not sevelamer. The aortic calcium content was significantly reduced by CaMg but not by sevelamer. The percent calcified area of the aorta was significantly lower than vehicle-treated animals for all three groups. The presence of aortic calcification was associated with increased sox9, bmp-2, and matrix gla protein expression, but this did not differ in the treatment groups. Calcium content in the carotid artery was lower with sevelamer than with CaMg but that in the femoral artery did not differ between groups. Thus, treatment with either CaMg or sevelamer effectively controlled serum phosphate levels in CRF rats and reduced aortic calcification.

Cell biological and physicochemical aspects of arterial calcification

Processes similar to endochondral or intramembranous bone formation occur in the vascular wall. Bone and cartilage tissue as well as osteoblast- and chondrocyte-like cells are present in calcified arteries. As in bone formation, apoptosis and matrix vesicles play an important role in the initiation of vascular calcification. Recent evidence indicates that nanocrystals initially formed in the vessel wall may actively be involved in the progression of the calcification process. This review focuses on the cellular and structural similarities between bone formation and vascular calcification and discusses the initial events in this pathological mineralization process.

Iron and vascular calcification. Is there a link

Iron deficiency is frequently seen in patients with end-stage renal disease, particularly in those treated by dialysis, this is because of an impairment in gastrointestinal absorption and ongoing blood losses or alternatively, due to an impaired capacity to mobilize iron from its stores, called functional iron deficiency. Therefore, these patients may require intravenous iron to sustain adequate treatment with erythropoietin-stimulating agents. Aside from this, they are also prone to vascular calcification, which has been reported a major contributing factor in the development of cardiovascular disease and the increased mortality associated herewith. Several factors and mechanisms underlying the development of vascular calcification in chronic kidney diseased patients have been put forward during recent years. In view of the ability of iron to exert direct toxic effects and to induce oxidative stress on the one hand versus its essential role in various cellular processes on the other hand, the possible role of iron in the development of vascular calcification should be considered.

Vascular Calcification Is Associated with Cortical Bone Loss in Chronic Renal Failure Rats with and without Ovariectomy: The Calcification Paradox

Background: Increased bone loss has been associated with the development of vascular calcification in patients with chronic renal failure (CRF). In this study, the effect of impaired bone metabolism on aortic calcifications was investigated in uremic rats with or without ovariectomy. Methods: CRF was induced by administration of a 0.75% adenine/2.5% protein diet for 4 weeks. In one group, osteoporosis was induced by ovariectomy (CRF-OVX), while the other group underwent a sham-operation instead (CRF). A third group consisted of ovariectomized rats with normal renal function (OVX). At regular time intervals throughout the study, bone status and aortic calcifications were evaluated by in vivo micro-CT. At sacrifice after 6 weeks of CRF, bone histomorphometry was performed and vascular calcification was assessed by bulk calcium analysis and Von Kossa staining. Results: Renal function was significantly impaired in the CRF-OVX and CRF groups. Trabecular bone loss was seen in all groups. In the CRF-OVX and CRF groups, trabecular bone density was restored after adenine withdrawal, which coincided with cortical bone loss and the development of medial calcifications in the aorta. No significant differences with regard to the degree of aortic calcifications were seen between the two CRF groups. Neither cortical bone loss nor calcifications were seen in the OVX group. Cortical bone loss significantly correlated with the severity of vascular calcification in the CRF-OVX and CRF groups, but no associations with trabecular bone changes were found. Conclusions: Cortical rather than trabecular bone loss is associated with the process of calcification in rats with adenine- induced CRF.

A Magnesium Based Phosphate Binder Reduces Vascular Calcification without Affecting Bone in Chronic Renal Failure Rats

The alternative phosphate binder calcium acetate/magnesium carbonate (CaMg) effectively reduces hyperphosphatemia, the most important inducer of vascular calcification, in chronic renal failure (CRF). In this study, the effect of low dose CaMg on vascular calcification and possible effects of CaMg on bone turnover, a persistent clinical controversy, were evaluated in chronic renal failure rats. Adenine-induced CRF rats were treated daily with 185 mg/kg CaMg or vehicle for 5 weeks. The aortic calcium content and area% calcification were measured to evaluate the effect of CaMg. To study the effect of CaMg on bone remodeling, rats underwent 5/6th nephrectomy combined with either a normal phosphorus diet or a high phosphorus diet to differentiate between possible bone effects resulting from either CaMg-induced phosphate deficiency or a direct effect of Mg. Vehicle or CaMg was administered at doses of 185 and 375 mg/kg/day for 8 weeks. Bone histomorphometry was performed. Aortic calcium content was significantly reduced by 185 mg/kg/day CaMg. CaMg ameliorated features of hyperparathyroid bone disease. In CRF rats on a normal phosphorus diet, the highest CaMg dose caused an increase in osteoid area due to phosphate depletion. The high phosphorus diet combined with the highest CaMg dose prevented the phosphate depletion and thus the rise in osteoid area. CaMg had no effect on osteoblast/osteoclast or dynamic bone parameters, and did not alter bone Mg levels. CaMg at doses that reduce vascular calcification did not show any harmful effect on bone turnover.

Marginal growth increase, altered bone quality and polycystic ovaries in female prepubertal rats after treatment with the aromatase inhibitor exemestane.

Background: Aromatase inhibition has been proposed as a potential approach for growth enhancement in children with short stature, but detailed animal studies are lacking. Aim: To assess the effect and potential adverse effects of aromatase inhibition on growth in female rats. Methods: Prepubertal Wistar rats received intramuscular injections with placebo or the aromatase inhibitor exemestane at a dose of 10, 30 or 100 mg/kg/week (E10, E30, E100) for 3 weeks. A control group was ovariectomized (OVX). Weight and length gain, tibia and femur length, growth plate width, organ weights, insulin-like growth factor I (IGF-I) levels, and histology of the ovaries, uterus and brain were analyzed. X-ray microtomography of femora was performed. Results: E100 significantly increased weight gain and growth plate width, but less prominently than OVX. Trabecular number and thickness were decreased in E100 and OVX in the metaphysis and epiphysis. E100 significantly decreased ovarian weight and multiple cysts were seen upon histological evaluation. No significant effects were found on IGF-I levels and brain morphology in E100. E10 and E30 had no effects on growth. Conclusion: A high dose of exemestane marginally increases axial and appendicular growth in female rats, at the expense of osteopenia and polycystic ovaries.

Impaired Body Weight and Tail Length Gain and Altered Bone Quality after Treatment with the Aromatase Inhibitor Exemestane in Male Rats

Background: Estrogen deficiency induced by aromatase inhibitors may be a novel treatment modality for growth enhancement in short children, but may have adverse effects on bone, brain and reproduction. Aim: To assess growth effects and potential adverse effects of aromatase inhibition in male rats. Methods: 26-day-old prepubertal rats received intramuscular injections with placebo or the aromatase inhibitor exemestane at a dose of 10, 30 or 100 mg/kg/week [E10, E30, E100(6)] for 6 weeks, completely covering the sexual maturation phase, or with 3 weeks E100 followed by 3 weeks placebo [E100(3)]. Growth parameters and histology of the testis, seminal vesicle and brain were analyzed. Bone architecture was studied with X-ray microtomography. Results: Exemestane dose-dependently decreased body weight and tail length gain, as well as liver and seminal vesicle weights, but did not affect nose-anus length gain, growth plate width or radial growth. E100(6) decreased trabecular thickness (epiphysis and metaphysis) and number (metaphysis). Normal IGF-I levels and brain, testis and seminal vesicle morphology were observed. E100(3) resulted in decreased tail length gain only. Conclusion: Exemestane treatment during sexual maturation did not augment linear growth in male rats, but caused impaired body weight and tail length gain and osteopenia.