Breast arterial calcification in chronic kidney disease: absence of smooth muscle apoptosis and osteogenic transdifferentiation

http://www.kidney-international.org & 2013 International Society of Nephrology see commentary on page 501 Breast arterial calcification in chronic kidney disease: absence of smooth muscle apoptosis and osteogenic transdifferentiation W. Charles O Neill 1 and Amy L. Adams 2 1 Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA and 2 Department of Pathology, Emory University School of Medicine, Atlanta, Georgia, USA The pathophysiology of medial arterial calcification in chronic kidney disease (CKD) is unclear but has been ascribed to phenotypic changes in vascular smooth muscle, possibly in conjunction with intimal proliferation and atherosclerosis. As the prevalence of calcification in breast arteries is increased in women with CKD and end-stage renal disease (ESRD), this was examined histologically in mastectomy specimens from 19 women with CKD or ESRD. Arterial calcification was present in 18, was exclusively medial, and occurred in vessels as small as arterioles. Intimal thickening was common but unrelated to calcification. There was no evidence of atherosclerosis. The earliest calcification presented as small punctate lesions scattered throughout the media, often with calcification of the internal elastic lamina. Arterial calcification was present in all samples from an age- and diabetesmatched cohort without CKD but was much milder. While smooth muscle cell density was reduced one-third in arteries from patients with ESRD, the cells appeared normal, expressed SM22a, and exhibited no apoptosis. Staining for the bone-specific protein osteocalcin, the osteoblastic transcription factors Runx2 or osterix, or the chondrocytic transcription factor SOX9 was absent in regions of early calcification. Thus, medial calcification in breast arteries of patients with CKD can occur in the absence of smooth muscle cell apoptosis and/or osteogenic transdifferentiation. This suggests that the pathologic mineralization process may differ from one arterial type to the other. Kidney International (2014) 85, 668 676; doi:10.1038/ki.2013.351; published online 18 September 2013 KEYWORDS: arteriosclerosis; vascular calcification; vascular smooth muscle Correspondence: W. Charles O Neill, Renal Division, Department of Medicine, Emory University School of Medicine, WMB 338, 1639 Pierce Drive, Atlanta, Georgia 30322, USA. E-mail: woneill@emory.edu Received 6 December 2012; revised 25 June 2013; accepted 27 June 2013; published online 18 September 2013 Calcification of the medial layer of arteries is an age-related lesion that is accelerated in chronic kidney disease (CKD), diabetes, and rare genetic disorders. The resulting stiffening of the arterial wall is thought to contribute to the high burden of cardiovascular disease in these patients. 1,2 Although this medial calcification appears histologically distinct from the intimal calcification that occurs in atherosclerosis, 3,4 the two forms often coincide and have similar risk factors and effects on outcomes, leading some to hypothesize that they are related. 5 Individuals with advanced renal failure are at a particular risk for medial arterial calcification, with a prevalence as much as fourfold greater than that in age-matched and diabetes-matched individuals without renal failure. 6,7 Current preventative measures are limited to control of circulating phosphate levels but are not applicable to individuals without kidney disease. Thus, there is a need for additional therapies that require a better understanding of the pathophysiology. Calcification of the smooth muscle matrix is a thermodynamically favored event that is normally prevented by inhibitors of hydroxyapatite formation. Elastin spontaneously calcifies in vitro and in vivo at physiologic calcium and phosphate concentrations, 8,9 and the genetic absence of the two principal endogenous inhibitors, pyrophosphate or matrix gla protein, results in medial vascular calcification in mice 10,11 and in humans. 12 14 Whether deficiency of these inhibitors underlies other cases of medial calcification is not known, although pyrophosphate levels are reduced in patients with end-stage renal disease (ESRD) 15 and correlate inversely with vascular calcification in patients with CKD and ESRD, 16 and exogenous pyrophosphate inhibits calcification in uremic rats. 17 Levels of carboxylated matrix gla protein may also be altered in calcified arteries. 18 As pyrophosphate is also a critical determinant of bone formation, 19,20 there may be important parallels between medial vascular calcification and osteogenesis. It is widely believed that transdifferentiation of smooth muscle cells into osteoblastic cells is the initial event in medial calcification, based on studies in cultured vascular smooth muscle cells and histologic observations in vessels from patients with CKD or ESRD. 21 23 However, cultured 668 Kidney International (2014) 85, 668 676

Table 1 Patient characteristics Patient Age (years) Race ESRD duration (years) Diabetes Serum creatinine (mg/ml) egfr (ml/min per 1.76 m 2 ) Serum calcium (mg/dl) Serum phosphorus (mg/dl) 1 66 AA Yes 1.2 53 9.4 2 87 W Yes 1.8 29 9.3 3 84 Unknown No 1.8 29 9.8 4 59 AA No 1.9 33 9.7 4.5 5 70 W No 2.0 29 9.2 6 48 AA No 2.1 31 9.4 3.2 7 69 AA No 2.1 27 9.2 8 84 W No 3.1 15 9.5 9 72 AA No 3.9 13 9 10 83 AA 0 Yes 8.2 4.9 11 51 AA 1.5 No 8.9 12 68 AA 2 Yes 8.4 4.1 13 53 W 3 Yes 8.4 5.2 14 65 AA 43.5 No 9.5 15 70 AA 5 Yes 8.8 16 64 W 5.5 Yes 8.8 4.8 17 62 W 6 Yes 9.7 18 26 AA 7 No 8.1 5.1 19 61 AA 7 Yes 9.2 Abbreviations: AA, African American; egfr, estimated glomerular filtration rate; ESRD, end-stage renal disease; W, white. cells are phenotypically quite different from normal smooth muscle and do not reflect conditions in vivo, and histologic studies have been limited to single, medium to-large intraabdominal arteries. An additional hypothesis that has been advanced is that renal failure induces apoptosis of smooth muscle cells that provide a nidus for calcification. 24 However, apoptosis or loss of smooth muscle cells is not a consistent finding 25 27 and may be a reaction to the calcification. 28 Thus, the possibility that phenotypic changes in vascular smooth muscle are a secondary event or occur only in certain arteries cannot be excluded. We have previously demonstrated that the prevalence of breast arterial calcification is increased in CKD. 7 As atherosclerosis does not occur in these vessels 29 and the calcification is exclusively medial, 6 specimens from breast excisions provided the opportunity to examine specifically the histology of medial calcification. Furthermore, the presence of multiple vessels in each specimen with a spectrum of calcification ranging from very early to advanced lesions provided the opportunity to define both the natural history of medial calcification and the role of phenotypic changes in smooth muscle. RESULTS A total of 19 patients were identified, of whom 10 had ESRD and 9 had CKD at the time of surgery. Surgery was performed for cancer in all cases and additional patient characteristics are shown in Table 1. The patients with CKD had serum creatinine values ranging from 1.2 to 3.9 mg/dl, and the ESRD patients were all undergoing hemodialysis. Serum calcium was lower in the ESRD patients and below the normal range in four patients. Serum phosphorus was not obtained in most patients. of the patients were receiving warfarin. Arterial calcification was detectable by hematoxylin and eosin staining and von Kossa staining in 14 specimens and only by von Kossa staining in 4 specimens. No calcification was observed in one specimen (Table 2). Calcification was more severe in the diabetic patients, with 100% of arteries involved in seven of nine and o50% involvement in only one. By comparison, only 1 of 10 nondiabetic patients exhibited calcification in all arteries, whereas 4 patients showed o50% involvement. The calcification was exclusively medial, and two patterns of staining were noted. Punctate staining distributed throughout the media (Figure 1a) was present in all specimens and was often concentrated at the intimal border. Much of this staining was at some distance from the nuclei, suggesting that it was extracellular. In half of the specimens, there was also linear staining of the internal elastic lamina (IEL). Although this was occasionally the predominant pattern (Figure 1b), it always coincided with punctate medial staining (Figure 1c). In more heavily calcified arteries, the IEL and subintimal calcification coalesced to form large confluent calcifications (Figure 1d). Linear IEL calcification was less frequent in the diabetic specimens than in the nondiabetic specimens (33% vs. 60%) but the difference was not significant. Calcification was frequently focal, with uninvolved arteries adjacent to calcified arteries (Figure 1e). Vessels of all sizes were affected, ranging from 1 1.5 mm to as small as 10 15 mm in luminal diameter (Figure 1f). Although intimal hyperplasia was present in some arteries, no atheromatous changes such as lipid-laden macrophages (foam cells), cholesterol clefts, or lipid pools were noted in any arteries in any specimen. Medial calcification occurred both in the presence (Figure 1c) and absence (Figure 1b) of intimal thickening. In a control group of 19 patients with serum creatinine o1.0 mg/dl (mean 0.81) and matched for age and diabetes with the CKD/ESRD cohort, medial calcification was present in all 15 specimens containing arteries. However, the Kidney International (2014) 85, 668 676 669

Table 2 Prevalence and pattern of medial calcification in breast arteries Specimen H&E VK % Calcified Diameter (lm) Pattern 1 Yes Yes 100 140 Linear IEL; punctate medial; confluent 2 No No 0 No calcifications 3 Yes Yes 100 190 Linear IEL; punctate medial 4 No Yes 50 75 170 Linear IEL; punctate medial 5 Yes 50 75 210 Punctate medial; confluent 6 Yes o25 650 Linear IEL; punctate medial 7 Yes Yes 475 1120 Linear IEL; punctate medial; confluent 8 Yes 475 110 Punctate medial 9 Yes Yes 475 130 Linear IEL; punctate medial 10 No Yes 100 460 Linear IEL; punctate medial 11 Yes o25 290 Linear IEL; punctate medial 12 Yes Yes 100 1060 Linear IEL; punctate medial 13 Yes Yes 100 450 Punctate medial 14 No Yes o25 180 Punctate medial 15 Yes 100 600 Punctate medial 16 Yes 100 195 Punctate medial 17 Yes 475 250 Punctate medial 18 Yes Yes o25 1320 Punctate medial; confluent 19 No Yes 100 135 Punctate medial Abbreviations: H&E, hematoxylin and eosin; IEL, internal elastic lamina; VK, von Kossa stain. Diameter indicates the size of the largest calcified artery and includes the media, intima, and the lumen. L calcification was much milder and apparent only by the von Kossa staining in all but one, with no confluent areas. Diffuse punctate calcification of the media was present in all 15 specimens, whereas linear calcification of the IEL was present in only 6. The smooth muscle cells appeared normal in calcified arteries, even when adjacent to heavy calcifications (Figure 2a). However, cell density within the media was significantly lower in nine ESRD specimens than in nine non- CKD specimens matched for age and diabetes (0.842± 0.08510 6 /mm 3 vs. 1.26±0.1410 6 /mm 3 ; P ¼ 0.022). This one-third reduction was identical to that previously observed in omental arteries from children with ESRD. 23 Essentially, all cells within the media of all arteries examined stained for SM22a, a marker of differentiated smooth muscle cells, even in the areas of heavy calcification (Figure 2b). Glandular epithelial cells did not take up the stain, demonstrating the specificity of the antibody (Figure 2c). The absence of apoptosis was confirmed by terminal deoxynucleotidyl transferase dutp nick end labeling (TUNEL) staining that was performed in three specimens containing arteries with substantial calcification and showed no medial staining including in heavily calcified regions (Figures 2d and e). Staining was apparent in areas of neointimal proliferation (Figure 2f) and in a specimen of lymphoma used as a positive control (not shown). Evidence for osteogenic transdifferentiation in smooth muscle cells was sought by immunostaining for the bonespecific protein osteocalcin and the osteoblastic transcription Figure 1 Patterns of calcification in breast arteries shown by von Kossa staining. (a) Diffuse punctate staining in the medial layer (patient 17). L indicates lumen. (b) Linear calcification of the internal elastic lamina (IEL) in patient 1. There is no intimal thickening. (c) Both diffuse medial and linear IEL calcification in the same artery (patient 12). Intimal thickening is present. (d) Severe confluent calcification (patient 1). (e) Two adjacent arteries with and without calcification (patient 8). (f) Calcification of small arteries and arterioles (patient 8). Bar ¼ 100 mm in all images. factor Runx2, as well as the loss of staining for SM22a. Figure 3 shows sequential sections of arteries with early lesions from two different specimens stained for calcium (von Kossa), osteocalcin, Runx2, and SM22a. Despite the presence of calcification, there was no staining for osteocalcin or Runx2, and the smooth muscle cells all stained for SM22a. Similar results are demonstrated in a larger, more heavily calcified artery (Figure 4). The results for all the specimens are presented in Table 3. Staining for osteocalcin was detected in half of the 18 specimens that were examined but only in more heavily calcified arteries. This staining was always extracellular and appeared to coincide with calcium deposits (Figures 5a and b), in contrast to the cellular staining of osteocytes within the bone (Figure 6a). This staining was not observed when the primary antibody was omitted. Staining for Runx2 was not observed in any calcified vessels, including heavily calcified vessels (Figures 3 and 4), but was apparent in placental tissue 670 Kidney International (2014) 85, 668 676

Patient 8 Patient 19 C L Figure 2 Histology of smooth muscle cells in calcified arteries. (a) Hematoxylin and eosin staining of a heavily calcified artery from patient 1 showing normal-appearing smooth muscle cells. (b) Staining for SM22a in the same artery. There was fracturing of the artery during sectioning. (c) Ductal epithelial cells do not stain for SM22a, whereas underlying smooth muscle is positive. (d) Terminal deoxynucleotidyl transferase dutp nick end labeling (TUNEL) staining of a large, heavily calcified artery from patient 18 demonstrating no apoptosis adjacent to the calcifications (C). There is some staining of cells in the adventitia (arrows) and some artifactual staining of the calcifications. Most of the calcification was lost during sectioning. L indicates the lumen. (e) The von Kossa staining pattern corresponding to the region shown in d. (f) Positive TUNEL staining of neointimal cells. Bars ¼ 100 mm. used as a positive control (Figure 6b). Osteocalcin staining was twice as frequent in the diabetic specimens (67% vs. 33%), probably reflecting greater calcification. To confirm the absence of osteogenic transdifferentiation, a subset of 12 specimens was also stained for osterix, a more specific osteoblastic transcription factor. Staining for osterix was absent in vascular smooth muscle cells within the calcified vessels (Figure 5c and d) but was present in the neonatal spine used as a positive control (Figure 6c). There was some mild, nonspecific staining that included the calcifications. Analysis of staining for SOX9, a chondrogenic marker, was hampered by nonspecific staining, but lack of cellular staining in calcified vessels was noted in at least two Kidney International (2014) 85, 668 676 Figure 3 Immunohistochemical analysis of matched sections of small breast arteries from two patients. (a) There is calcification within the media and of the internal elastic lamina (von Kossa staining). (b) All cells within the media stained for SM22a. There was no staining for osteocalcin (c) or Runx2 (d). specimens (Figures 5e and f). Staining was noted in chrondroblasts from neonatal mouse spine (Figure 6d). For comparison, immunohistochemical analysis was also performed on large, heavily calcified arteries (seven tibial and three femoral) from amputations of lower limbs in patients with ESRD. Again, there was frequent staining of the calcifications for osteocalcin, but cellular staining was apparent in 3 of the 10 specimens from different patients 671

Table 3 Immunohistochemical analysis of calcified breast arteries Specimen Osteocalcin staining Runx2 staining 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 ND ; no VK match Yes ; no VK match ND ; no VK match ; no VK match Abbreviations: ND, not done; VK, von Kossa stain. DISCUSSION Figure 4 Immunohistochemical analysis of matched sections of a large breast artery from patient 12. Left hand column: low magnification; bars ¼ 100 mm. Right hand column: higher magnification of region indicated by box. (a) The von Kossa staining. (b) SM22a. (c) Osteocalcin. (d) Runx2. Bars ¼ 100 mm. There is no staining for osteocalcin or Runx2. (Figures 7a and b). In two of these, the stained cells were within calcifications that were also stained, so that differentiation between cellular and extracellular staining was not certain. Three specimens (one tibial and two femoral) were also stained for Runx2 (Figure 7c) or osterix (Figure 7d). Medial staining was absent but there was occasional staining for Runx2 and osterix in the intima in the absence of any calcification. 672 The presence in breast tissue of a wide range of arteries with variable involvement provides a unique opportunity to define the natural history and pathophysiology of medial arterial calcification. The findings confirm that vascular calcification in breast arteries in CKD is exclusively medial, consistent with studies in the general population.29 Medial arterial calcification was also found in all of the specimens from control patients without CKD. However, the calcification was substantially milder, suggesting that renal insufficiency or its associated alterations in mineral metabolism are not necessary initiating events in medial calcification but instead exacerbate an underlying tendency of these arteries to calcify. The absence of intimal calcification can be explained by the absence of atherosclerosis in breast arteries, as noted in the aforementioned study in the general population29 and confirmed in CKD patients in this study. Specifically, there were no cholesterol clefts or lipid pools and no inflammatory changes in the intima. Although intimal hyperplasia was noted in a number of arteries, there was no relationship to medial calcification, and extensive medial calcification was observed in numerous arteries without intimal hyperplasia. Furthermore, calcification was seen in arteries as small as arterioles, which are not affected by atherosclerosis.30 This provides further evidence that intimal and medial calcifications are distinct lesions. Although our study is limited to breast tissue in women, the results are likely indicative of the effect of renal failure in other arteries and in men, as breast arterial calcification is increased in CKD7 and correlates with calcification in peripheral arteries.6 Two basic patterns of calcification were noted: diffuse punctate calcification, often concentrated in the subintimal area and around the IEL, and linear calcification of the IEL. Kidney International (2014) 85, 668 676

Figure 6 Positive controls for immunohistochemical analysis. (a) Sample of bone showing cellular staining of the osteocytes for osteocalcin. (b) Runx2 staining of placenta. (c) Osterix immunostaining of neonatal mouse spine. (d) SOX-9 immunostaining of neonatal mouse spine. Bars ¼ 100 mm. I M Figure 5 Staining for osteogenic/chondrogenic markers. (a) Staining for osteocalcin in patient 17 showing a granular pattern within the media and in the internal elastic lamina. (b) Osteocalcin staining of heavy calcifications in patient 18. The vessel wall has fractured during sectioning. (c) The von Kossa staining of artery from patient 13. (d) Osterix immunostaining of the same artery. (e) The von Kossa staining of artery from patient 12. (f) SOX-9 immunostaining of the same artery. Bars ¼ 100 mm. Calcification of the IEL has been variably described in other reports (reviewed in ref. 23), and its relationship to calcification within the media has been debated. Some investigators have proposed that IEL calcification occurs in all cases and is the initial event.31 However, the lesions examined in that study were relatively advanced, and the authors noted the need to study a range of lesions to determine the natural history. Although IEL calcification was clearly present in breast arteries, calcification frequently occurred in its absence, and it is clear that the earliest lesion is punctate staining within the media. However, it was also clear from examination of lesions with varying severity that it is the calcification of the IEL and subintimal media that progresses into the confluent areas of calcification noted in advanced lesions. Although varying degrees of diffuse punctate calcification were noted within the media, these never appeared to become confluent. The frequent and Kidney International (2014) 85, 668 676 M A I M M A A Figure 7 Osteogenic markers in calcified arteries from lower extremities. Arrows indicate the internal elastic lamina with the intima (I) to the right, the media (M) to the left, and the adventitia (A) to the far left. (a) Decalcified section of a tibial artery showing staining for osteocalcin of the medial calcifications and some smooth muscle cells within the calcifications. (b) Section of a tibial artery showing no staining for osteocalcin. The artery fractured at the calcifications during sectioning, with loss of the intima. (c) Decalcified section of a tibial artery showing no staining for Runx2. The intima was lost due to a fracture at the internal elastic lamina during sectioning. (d) Decalcified section of a femoral artery from a patient with chronic kidney disease showing no staining for osterix. Bars ¼ 100 mm. 673

prominent calcification of the IEL indicates that calcification can be an extracellular process that does not necessarily begin within cells. Although the location of the punctate medial staining cannot be determined with certainty, the pattern is also suggestive of extracellular calcification. A surprising finding was the extent of involvement of small arteries, including small arterioles. There did not appear to be any relationship between calcification and vessel size, and arteries of all sizes were affected in most specimens. Medial calcification is generally considered to occur in large and medium muscular arteries and, therefore, to affect hemodynamics through decreased compliance of conduit vessels. 1 However, the involvement of resistance vessels shown in this study could lead to additional effects such as hypertension and tissue ischemia due to impaired vasodilation. An additional finding was the normal appearance of smooth muscle cells within and adjacent to areas of calcification and with uniform staining for SM22a. Previous studies have suggested that medial calcification is associated with loss of smooth muscle cells and may begin in apoptotic cells. 23,32 Although the density of medial smooth muscle cells was decreased in breast arteries from ESRD patients, similar to prior results in omental arteries, 23 there was no obvious cell dropout or evidence of apoptosis, even in more advanced lesions. This is consistent with a previous study showing a normal appearance of smooth muscle cells with heavy expression of SM22a adjacent to advanced medial calcifications in peripheral arteries. 25 Most likely, apoptosis of smooth muscle cells is a late event in medial calcification, possibly due to a toxic effect of calcium phosphate crystals 28 and is unrelated to its initiation. The absence of apoptosis suggests that the decreased cell density likely represents an increase in smooth muscle matrix, as previously demonstrated, 33 rather than cell loss. The results in breast arteries were notable for the lack of evidence of osteogenic transdifferentiation of vascular smooth muscle cells. Although staining for osteocalcin, a bone-specific protein, was observed in some vessels in half of the specimens, it was always extracellular in areas of heavy calcification and coincided with the calcifications. Although this could represent binding of the antibody to the calcifications, it likely represents binding of circulating osteocalcin to existing calcifications, consistent with the high affinity of osteocalcin for hydroxyapatite. 34 Such binding has been demonstrated in devitalized heart valves that calcified after implantation in vivo, even within a barrier that prevented influx of cells. 35 Staining for Runx2 or osterix was never observed. The presence of substantial nonspecific staining for the chondrogenic factor SOX9 prevented any firm conclusions, but the absence of SOX9 was documented in calcified arteries in at least two specimens. These results indicate that medial calcification can occur in the absence of osteogenic transdifferentiation. Although it is possible that calcification in breast arteries is a different process than medial arterial calcification in other tissues, this seems unlikely as breast arterial calcification in ESRD patients, as judged by mammography, correlates with a pattern of medial calcification in radiographs of extremities. 6 The possibility that the pathophysiology of medial calcification may differ between the arterial types was addressed by repeating immunohistochemical analysis in large, heavily calcified arteries from amputation specimens. Aside from some staining for osteocalcin in a minority of arteries, there was no evidence of osteogenic transdifferentiation, raising questions about the role of this putative process in general. Previous studies have relied on staining for tissue-nonspecific alkaline phosphatase, osteopontin, or Runx2, but tissue-nonspecific alkaline phosphatse and osteopontin are widely distributed among tissues and Runx2 is expressed in a number of other cell types. 36 Thus, none is a specific marker of osteogenic cells. Although more selective markers such as osterix and bone sialoprotein have been used, specificity remains an issue. Staining has often appeared noncellular, as was the case in the current study, possibly representing binding of antibodies to calcifications. Although cellular osterix staining has been noted in calcified arteries, 23 it was also present in normal arteries. In the current study, careful titration of the antibodies with positive and negative controls was required to minimize this nonspecific staining. In summary, the results demonstrate that medial calcification in breast arteries occurs in the absence of any apparent phenotypic change in vascular smooth muscle, indicating that apoptosis or osteogenic transdifferentiation are not necessary initiating events for medial calcification. If these events are in fact involved in medial calcification in other vessels, then the results further demonstrate that the pathogenesis of medial calcification differs between the arterial types. MATERIALS AND METHODS Patients A computerized search of medical records was performed to identify women who had undergone mastectomy, partial mastectomy, or lumpectomy from 2007 to 2011 at Emory Healthcare and who also carried a diagnosis of ESRD or CKD. Charts were reviewed to confirm the diagnosis of ESRD or CKD and to exclude patients with acute renal failure or transplantation. The serum creatinine values used to determine the stage of CKD were obtained within 7 days of surgery in all but two patients in whom they were obtained within 5 months. A control cohort of mastectomy patients without renal insufficiency (serum creatinine o1.0 mg/dl) was selected to match the age and diabetes status of the CKD/ESRD patients. Samples Surgical specimens were processed in the Clinical Pathology department, fixed in formalin, and embedded in paraffin. The original specimen slides stained with hematoxylin and eosin were reviewed for the presence of arteries. Corresponding paraffin blocks were retrieved and additional slides were prepared from multiple, sequential 5-mm-thick sections. 674 Kidney International (2014) 85, 668 676

Histologic analysis Staining for calcification was performed by the von Kossa method using a standard clinical protocol. Staining for apoptosis was performed by TUNEL with fluorescein (Roche Diagnostics, Indianapolis, IN), according to the instructions from the vendor. The following antibodies were used for immunohistochemical analysis: anti-runx2 (27-K mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX), anti-osteocalcin (OC4-30 mouse monoclonal; Abcam, Cambridge, MA), anti-smooth muscle myosin 22a (goat polyclonal; Abcam), anti-osterix (rabbit polyclonal; Abcam), and anti-sox9 (rabbit polyclonal; Abcam). Staining was performed according to protocols supplied by the vendors with appropriate antibody dilutions determined on positive and negative controls. Human bone and placenta were used as positive controls for osteocalcin and Runx2, respectively, and neonatal mouse spine was the positive control for osterix and SOX9. Secondary antibodies were horseradish-linked anti-mouse (EnVision Dual Link, Dako, Carpenteria, CA) and anti-goat or anti-rabbit (Biocare Medical, Concord, CA). Analysis Sections stained with hematoxylin and eosin and by the von Kossa method were examined in their entirety to assess the range and pattern of calcification and the size of vessels affected. Sections contained a minimum of 10 arteries and usually many more. All arteries were also examined in sections stained for apoptosis or immunohistochemical analysis. Selected vessels were matched with identical vessels located on the von Kossa stained slides to compare staining and calcification in identical regions in identical arteries. There was at least one, and usually three, matching arteries for each specimen, although matching was not possible for some of the stains in a few instances. Cell density within the media was assessed by tracing the medial layer on 400 images of sections stained with hematoxylin and eosin, counting the nuclei within, and dividing by the tissue volume (surface areathe 5 mm thickness). Only arteries with at least four layers of smooth muscle cells were analyzed and regions of confluent calcification were not included. Significance was determined by two-tailed t-testing or by w 2 testing for categorical variables. DISCLOSURE All the authors declared no competing interests. ACKNOWLEDGMENTS This study was supported by a Grant-in-Aid from the American Heart Association. REFERENCES 1. Blacher J, Guerin AP, Pannier B et al. Arterial calcifications, arterial stiffness, and cardiovascular risk in end-stage renal disease. Hypertension 2001; 38: 938 942. 2. London GM, Guerin AP, Marchais SJ et al. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant 2003; 18: 1731 1740. 3. Jeziorska M, McCollum C, Woolley DE. Calcification in atherosclerotic plaque of human carotid arteries: associations with mast cells and macrophages. J Pathol 1998; 185: 10 17. 4. Amann K. Media calcification and intima calcification are distinct entities in chronic kidney disease. Clin J Am Soc Nephrol 2008; 3: 1599 1605. 5. McCullough PA, Agrawal V, Danielewicz E et al. Accelerated atherosclerotic calcification and Mönckeberg s sclerosis: a continuum of advanced vascular pathology in chronic kidney disease. Clin J Am Soc Nephrol 2008; 3: 1585 1598. 6. Duhn V, D Orsi EM, Johnson S et al. Breast arterial calcification: a marker of medial vascular calcification in chronic kidney disease. Clin J Am Soc Nephrol 2011; 6: 377 382. 7. Abou-Hassan N, D Orsi ET, D Orsi CJ et al. The risk for medial arterial calcification in CKD. Clin J Am Soc Nephrol 2012; 7: 275 279. 8. Urry DW. Neutral sites for calcium ion binding to elastin and collagen: a charge neutralization theory for calcification and its relationship to atherosclerosis. Proc Natl Acad Sci USA 1971; 68: 810 814. 9. Vyavahare N, Ogle M, Schoen FJ et al. Elastin calcification and its prevention with aluminum chloride pretreatment. Am J Pathol 1999; 155: 973 982. 10. Harmey D, Hessle L, Narisawa S et al. Concerted regulation of inorganic pyrophosphate and osteopontin by Akp2, Enpp1 and Ank: an integrated model of the pathogenesis of mineralization disorders. Am J Pathol 2004; 164: 1199 1209. 11. Luo G, Ducy P, McKee MD et al. Spontaneous calcification of arteries and cartilage in mice lacking matrix Gla protein. Nature 1997; 386: 78 81. 12. Rutsch F, Vaingankar S, Johnson K et al. PC-1 nucleotide triphosphate pyrophosphohydrolase deficiency in idiopathic infantile arterial calcification. Am J Pathol 2001; 158: 543 554. 13. Meier M, Weng LP, Alexandrakis E et al. Tracheobronchial stenosis in Keutel syndrome. Eur Respir J 2001; 17: 566 569. 14. Munroe PB, Olgunturk RO, Fryns JP et al. Mutations in the gene encoding the human matrix Gla protein cause Keutel syndrome. Nature Genetics 1999; 21: 142 144. 15. Lomashvili KA, Khawandi W, O Neill WC. Reduced plasma pyrophosphate levels in hemodialysis patients. J Am Soc Nephrol 2005; 16: 2495 2500. 16. O Neill WC, Sigrist MK, McIntyre CW. Plasma pyrophosphate and vascular calcification in chronic kidney disease. Nephrol Dial Transplant 2010; 25: 187 191. 17. O Neill WC, Lomashvili KA, Malluche HH et al. Treatment with pyrophosphate inhibits uremic vascular calcification. Kidney Int 2011; 79: 512 517. 18. Schurgers LJ, Teunissen KJF, Knapen MHJ et al. Novel conformationspecific antibodies against matrix g-carboxyglutamic acid (gla) protein. Undercarboxylated matrix gla protein as marker for vascular calcification. Arterioscler Thromb Vasc Biol 2005; 25: 1629 1633. 19. Murshed M, Harmey D, Millan JL et al. Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev 2005; 19: 1093 1104. 20. Hessle L, Johnsson KA, Anderson HC et al. Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization. Proc Natl Acad Sci USA 2002; 99: 9445 9449. 21. Shanahan CM, Cary NRB, Salisbury JR et al. Medial localization of mineralization-regulating proteins in association with Monckeberg s sclerosis. Evidence for smooth muscle cell-mediated vascular calcification. Circulation 1999; 100: 2168 2176. 22. Moe SM, Duan D, Doehle BP et al. Uremia induces the osteoblast differentiation factor Cbfa1 in human blood vessels. Kidney Int 2003; 63: 1003 1011. 23. Shroff RC, McNair R, Figg N et al. Dialysis accelerates medial vascular calcification in part by triggering smooth muscle cell apoptosis. Circulation 2008; 118: 1748 1757. 24. Reynolds JL, Joannides AJ, Skepper JN et al. Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. JAm Soc Nephrol 2004; 15: 2857 2867. 25. Shanahan CM, Cary NRB, Salisbury JR et al. Medial localization of mineralization-regulating proteins in association with Monckeberg s sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation 1999; 100: 2168 2176. 26. Speer MY, Yang H-Y, Brabb T et al. Smooth muscle cells give rise to osteochondrogenic precursors and chondrocytes in calcifying arteries. Circ Res 2009; 104: 733 741. 27. Neven E, Dauwe S, De Broe ME et al. Endochondral bone formation is involved in media calcification in rats and in men. Kidney Int 2007; 72: 574 581. 28. Ewence AE, Bootman M, Roderick HL et al. Calcium phosphate crystals induce cell death in human vascular smooth muscle cells: a potential mechanism in atherosclerotic plaque destabilization. Circ Res 2008; 103: e28 e34. Kidney International (2014) 85, 668 676 675

29. Nielsen BB, Holm NV. Calcification in breast arteries. The frequency and severity of arterial calcification in female breast tissue without malignant changes. Acta Path Microbiol Immunol Scand A 1985; 93: 13 16. 30. Moore S. Blood vessels and lymphatics. In: Damjanov I, Linder J (eds) Anderson s Pathology, 10th edn Mosby: St. Louis, 1996, pp 1401. 31. Micheletti RG, Fishbein GA, Currier JS et al. Monckeberg sclerosis revisited. A clarification of the histologic definition of Monckeberg sclerosis. Arch Pathol Lab Med 2008; 132: 43 47. 32. Proudfoot D, Skepper JN, Hegyi L et al. Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies. Circ Res 2000; 87: 1055 1062. 33. Amann K, Wolf B, Nichols C et al. Aortic changes in experimental renal failure. Hyperplasia or hypertrophy of smooth muscle cells? Hypertension 1997; 29: 770 775. 34. Romberg RW, Werness PG, Riggs BL et al. Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium-binding proteins. Biochem 1986; 25: 1176 1180. 35. Levy RJ, Schoen FJ, Levy JT et al. Biologic determinants of dystrophic calcification and osteocalcin deposition in glutaraldehyde-preserved porcine aortic valve leaflets implanted subcutaneously in rats. Am J Pathol 1983; 113: 143 155. 36. Cameron ER, Neil JC. The Runx genes: lineage-specific oncogenes and tumor suppressors. Oncogene 2004; 23: 4308 4314. 676 Kidney International (2014) 85, 668 676