Breast arterial calcification in chronic kidney disease: absence of smooth muscle apoptosis and osteogenic transdifferentiation
2013; Elsevier BV; Volume: 85; Issue: 3 Linguagem: Inglês
10.1038/ki.2013.351
ISSN1523-1755
AutoresW. Charles O’Neill, Amy L. Adams,
Tópico(s)Dermatological and Skeletal Disorders
ResumoThe 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 diabetes-matched 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 SM22α, 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. 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 diabetes-matched 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 SM22α, 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. 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.Blacher J. Guerin A.P. Pannier B. et al.Arterial calcifications, arterial stiffness, and cardiovascular risk in end-stage renal disease.Hypertension. 2001; 38: 938-942Crossref PubMed Scopus (1234) Google Scholar,2.London G.M. Guerin A.P. Marchais S.J. et al.Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality.Nephrol Dial Transplant. 2003; 18: 1731-1740Crossref PubMed Scopus (1509) Google Scholar Although this medial calcification appears histologically distinct from the intimal calcification that occurs in atherosclerosis,3.Jeziorska M. McCollum C. Woolley D.E. Calcification in atherosclerotic plaque of human carotid arteries: associations with mast cells and macrophages.J Pathol. 1998; 185: 10-17Crossref PubMed Scopus (118) Google Scholar,4.Amann K. Media calcification and intima calcification are distinct entities in chronic kidney disease.Clin J Am Soc Nephrol. 2008; 3: 1599-1605Crossref PubMed Scopus (281) Google Scholar the two forms often coincide and have similar risk factors and effects on outcomes, leading some to hypothesize that they are related.5.McCullough P.A. 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: 1585-1598Crossref PubMed Scopus (140) Google Scholar 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.Duhn V. D'Orsi E.M. Johnson S. et al.Breast arterial calcification: a marker of medial vascular calcification in chronic kidney disease.Clin J Am Soc Nephrol. 2011; 6: 377-382Crossref PubMed Scopus (62) Google Scholar,7.Abou-Hassan N. D'Orsi E.T. D'Orsi C.J. et al.The risk for medial arterial calcification in CKD.Clin J Am Soc Nephrol. 2012; 7: 275-279Crossref PubMed Scopus (38) Google Scholar 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.Urry D.W. 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: 810-814Crossref PubMed Scopus (156) Google Scholar,9.Vyavahare N. Ogle M. Schoen F.J. et al.Elastin calcification and its prevention with aluminum chloride pretreatment.Am J Pathol. 1999; 155: 973-982Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar and the genetic absence of the two principal endogenous inhibitors, pyrophosphate or matrix gla protein, results in medial vascular calcification in mice10.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: 1199-1209Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar,11.Luo G. Ducy P. McKee M.D. et al.Spontaneous calcification of arteries and cartilage in mice lacking matrix Gla protein.Nature. 1997; 386: 78-81Crossref PubMed Scopus (1748) Google Scholar and in humans.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: 543-554Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 13.Meier M. Weng L.P. Alexandrakis E. et al.Tracheobronchial stenosis in Keutel syndrome.Eur Respir J. 2001; 17: 566-569Crossref PubMed Scopus (96) Google Scholar, 14.Munroe P.B. Olgunturk R.O. Fryns J.P. et al.Mutations in the gene encoding the human matrix Gla protein cause Keutel syndrome.Nature Genetics. 1999; 21: 142-144Crossref PubMed Scopus (337) Google Scholar 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.Lomashvili K.A. Khawandi W. O'Neill W.C. Reduced plasma pyrophosphate levels in hemodialysis patients.J Am Soc Nephrol. 2005; 16: 2495-2500Crossref PubMed Scopus (150) Google Scholar and correlate inversely with vascular calcification in patients with CKD and ESRD,16.O'Neill W.C. Sigrist M.K. McIntyre C.W. Plasma pyrophosphate and vascular calcification in chronic kidney disease.Nephrol Dial Transplant. 2010; 25: 187-191Crossref PubMed Scopus (119) Google Scholar and exogenous pyrophosphate inhibits calcification in uremic rats.17.O'Neill W.C. Lomashvili K.A. Malluche H.H. et al.Treatment with pyrophosphate inhibits uremic vascular calcification.Kidney Int. 2011; 79: 512-517Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar Levels of carboxylated matrix gla protein may also be altered in calcified arteries.18.Schurgers L.J. Teunissen K.J.F. Knapen M.H.J. et al.Novel conformation-specific antibodies against matrix γ-carboxyglutamic acid (gla) protein. Undercarboxylated matrix gla protein as marker for vascular calcification.Arterioscler Thromb Vasc Biol. 2005; 25: 1629-1633Crossref PubMed Scopus (259) Google Scholar As pyrophosphate is also a critical determinant of bone formation,19.Murshed M. Harmey D. Millan J.L. et al.Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone.Genes Dev. 2005; 19: 1093-1104Crossref PubMed Scopus (482) Google Scholar,20.Hessle L. Johnsson K.A. Anderson H.C. 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: 9445-9449Crossref PubMed Scopus (679) Google Scholar 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.Shanahan C.M. Cary N.R.B. Salisbury J.R. 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: 2168-2176Crossref PubMed Google Scholar, 22.Moe S.M. Duan D. Doehle B.P. et al.Uremia induces the osteoblast differentiation factor Cbfa1 in human blood vessels.Kidney Int. 2003; 63: 1003-1011Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 23.Shroff R.C. McNair R. Figg N. et al.Dialysis accelerates medial vascular calcification in part by triggering smooth muscle cell apoptosis.Circulation. 2008; 118: 1748-1757Crossref PubMed Scopus (395) Google Scholar However, cultured 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 intra-abdominal 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.Reynolds J.L. Joannides A.J. Skepper J.N. 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.J Am Soc Nephrol. 2004; 15: 2857-2867Crossref PubMed Scopus (758) Google Scholar However, apoptosis or loss of smooth muscle cells is not a consistent finding25.Shanahan C.M. Cary N.R.B. Salisbury J.R. 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: 2168-2176Crossref PubMed Scopus (572) Google Scholar, 26.Speer M.Y. Yang H-Y. Brabb T. et al.Smooth muscle cells give rise to osteochondrogenic precursors and chondrocytes in calcifying arteries.Circ Res. 2009; 104: 733-741Crossref PubMed Scopus (431) Google Scholar, 27.Neven E. Dauwe S. De Broe M.E. et al.Endochondral bone formation is involved in media calcification in rats and in men.Kidney Int. 2007; 72: 574-581Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar and may be a reaction to the calcification.28.Ewence A.E. Bootman M. Roderick H.L. 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-e34Crossref PubMed Scopus (251) Google Scholar 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.Abou-Hassan N. D'Orsi E.T. D'Orsi C.J. et al.The risk for medial arterial calcification in CKD.Clin J Am Soc Nephrol. 2012; 7: 275-279Crossref PubMed Scopus (38) Google Scholar As atherosclerosis does not occur in these vessels29.Nielsen B.B. Holm N.V. 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: 13-16PubMed Google Scholar and the calcification is exclusively medial,6.Duhn V. D'Orsi E.M. Johnson S. et al.Breast arterial calcification: a marker of medial vascular calcification in chronic kidney disease.Clin J Am Soc Nephrol. 2011; 6: 377-382Crossref PubMed Scopus (62) Google Scholar 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. 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. None 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 <50% involvement in only one. By comparison, only 1 of 10 nondiabetic patients exhibited calcification in all arteries, whereas 4 patients showed 3.5No9.51570AA5Yes8.81664W5.5Yes8.84.81762W6Yes9.71826AA7No8.15.11961AA7Yes9.2Abbreviations: AA, African American; eGFR, estimated glomerular filtration rate; ESRD, end-stage renal disease; W, white. Open table in a new tab Table 2Prevalence and pattern of medial calcification in breast arteriesSpecimenH&EVK% CalcifiedDiameter (μm)Pattern1YesYes100140Linear IEL; punctate medial; confluent2NoNo0No calcifications3YesYes100190Linear IEL; punctate medial4NoYes50–75170Linear IEL; punctate medial5FaintYes50–75210Punctate medial; confluent6FaintYes 751120Linear IEL; punctate medial; confluent8FaintYes>75110Punctate medial9YesYes>75130Linear IEL; punctate medial10NoYes100460Linear IEL; punctate medial11FaintYes<25290Linear IEL; punctate medial12YesYes1001060Linear IEL; punctate medial13YesYes100450Punctate medial14NoYes 75250Punctate medial18YesYes<251320Punctate medial; confluent19NoYes100135Punctate medialAbbreviations: 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. Open table in a new tab Abbreviations: AA, African American; eGFR, estimated glomerular filtration rate; ESRD, end-stage renal disease; W, white. 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. 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 1–1.5mm to as small as 10–15μm 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 <1.0mg/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 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±0.085 × 106/mm3 vs. 1.26±0.14 × 106/mm3; P=0.022). This one-third reduction was identical to that previously observed in omental arteries from children with ESRD.23.Shroff R.C. McNair R. Figg N. et al.Dialysis accelerates medial vascular calcification in part by triggering smooth muscle cell apoptosis.Circulation. 2008; 118: 1748-1757Crossref PubMed Scopus (395) Google Scholar Essentially, all cells within the media of all arteries examined stained for SM22α, 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 bone-specific protein osteocalcin and the osteoblastic transcription factor Runx2, as well as the loss of staining for SM22α. Figure 3 shows sequential sections of arteries with early lesions from two different specimens stained for calcium (von Kossa), osteocalcin, Runx2, and SM22α. Despite the presence of calcification, there was no staining for osteocalcin or Runx2, and the smooth muscle cells all stained for SM22α. 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 used as a positive control (Figure 6b). Osteocalcin staining was twice as frequent in the diabetic specimens (67% vs. 33%), probably reflecting greater calcification.Figure 4Immunohistochemical analysis of matched sections of a large breast artery from patient 12. Left hand column: low magnification; bars=100μm. Right hand column: higher magnification of region indicated by box. (a) The von Kossa staining. (b) SM22α. (c) Osteocalcin. (d) Runx2. Bars=100μm. There is no staining for osteocalcin or Runx2.View Large Image Figure ViewerDownload (PPT)Table 3Immunohistochemical analysis of calcified breast arteriesSpecimenOsteocalcin stainingRunx2 staining1FaintNone2NDND3FaintNone4NoneNone5NoneNone6None; no VK matchNone7YesNone8FaintNone9None; no VK matchNone; no VK match10FaintNone11NoneNone; no VK match12NoneNone13FaintNone14NoneNone15NoneNone16FaintNone17FaintNone18NoneNone19FaintNoneAbbreviations: ND, not done; VK, von Kossa stain. Open table in a new tab Figure 5Staining 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=100μm.View Large Image Figure ViewerDownload (PPT)Figure 6Positive 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=100μm.View Large Image Figure ViewerDownload (PPT) Abbreviations: ND, not done; VK, von Kossa stain. 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 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 (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. 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.Nielsen B.B. Holm N.V. 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: 13-16PubMed Google Scholar 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.Nielsen B.B. Holm N.V. 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: 13-16PubMed Google Scholar 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.Moore S. Blood vessels and lymphatics.in: Damjanov I. Linder J. Anderson’s Pathology. 10th edn. Mosby, St. Louis1996: 1401Google Scholar 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.Abou-Hassan N. D'Orsi E.T. D'Orsi C.J. et al.The risk for medial arterial calcification in CKD.Clin J Am Soc Nephrol. 2012; 7: 275-279Crossref PubMed Scopus (38) Google Scholar and correlates with calcification in peripheral arteries.6.Duhn V. D'Orsi E.M. Johnson S. et al.Breast arterial calcification: a marker of medial vascular calcification in chronic kidney disease.Clin J Am Soc Nephrol. 2011; 6: 377-382Crossref PubMed Scopus (62) Google Scholar 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. Calcification of the IEL has been variably described in other reports (reviewed in ref. 23.Shroff R.C. McNair R. Figg N. et al.Dialysis accelerates medial vascular calcification in part by triggering smooth muscle cell apoptosis.Circulation. 2008; 118: 1748-1757Crossref PubMed Scopus (395) Google Scholar), 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.Micheletti R.G. Fishbein G.A. Currier J.S. et al.Monckeberg sclerosis revisited. A clarification of the histologic definition of Monckeberg sclerosis.Arch Pathol Lab Med. 2008; 132: 43-47Crossref PubMed Google Scholar 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 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.Blacher J. Guerin A.P. Pannier B. et al.Arterial calcifications, arterial stiffness, and cardiovascular risk in end-stage renal disease.Hypertension. 2001; 38: 938-942Crossref PubMed Scopus (1234) Google Scholar 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 SM22α. Previous studies have suggested that medial calcification is associated with loss of smooth muscle cells and may begin in apoptotic cells.23.Shroff R.C. McNair R. Figg N. et al.Dialysis accelerates medial vascular calcification in part by triggering smooth muscle cell apoptosis.Circulation. 2008; 118: 1748-1757Crossref PubMed Scopus (395) Google Scholar,32.Proudfoot D. Skepper J.N. Hegyi L. et al.Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies.Circ Res. 2000; 87: 1055-1062Crossref PubMed Scopus (581) Google Scholar Although the density of medial smooth muscle cells was decreased in breast arteries from ESRD patients, similar to prior results in omental arteries,23.Shroff R.C. McNair R. Figg N. et al.Dialysis accelerates medial vascular calcification in part by triggering smooth muscle cell apoptosis.Circulation. 2008; 118: 1748-1757Crossref PubMed Scopus (395) Google Scholar 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 SM22α adjacent to advanced medial calcifications in peripheral arteries.25.Shanahan C.M. Cary N.R.B. Salisbury J.R. 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: 2168-2176Crossref PubMed Scopus (572) Google Scholar Most likely, apoptosis of smooth muscle cells is a late event in medial calcification, possibly due to a toxic effect of calcium phosphate crystals28.Ewence A.E. Bootman M. Roderick H.L. 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-e34Crossref PubMed Scopus (251) Google Scholar 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.Amann K. Wolf B. Nichols C. et al.Aortic changes in experimental renal failure. Hyperplasia or hypertrophy of smooth muscle cells?.Hypertension. 1997; 29: 770-775Crossref PubMed Scopus (95) Google Scholar 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.Romberg R.W. Werness P.G. Riggs B.L. et al.Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium-binding proteins.Biochem. 1986; 25: 1176-1180Crossref PubMed Scopus (246) Google Scholar 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.Levy R.J. Schoen F.J. Levy J.T. 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: 143-155PubMed Google Scholar 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.Duhn V. D'Orsi E.M. Johnson S. et al.Breast arterial calcification: a marker of medial vascular calcification in chronic kidney disease.Clin J Am Soc Nephrol. 2011; 6: 377-382Crossref PubMed Scopus (62) Google Scholar 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.Cameron E.R. Neil J.C. The Runx genes: lineage-specific oncogenes and tumor suppressors.Oncogene. 2004; 23: 4308-4314Crossref PubMed Scopus (134) Google Scholar 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.Shroff R.C. McNair R. Figg N. et al.Dialysis accelerates medial vascular calcification in part by triggering smooth muscle cell apoptosis.Circulation. 2008; 118: 1748-1757Crossref PubMed Scopus (395) Google Scholar 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. 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 <1.0mg/dl) was selected to match the age and diabetes status of the CKD/ESRD patients. 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-μm-thick sections. 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). 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 area × the 5μm 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 χ2 testing for categorical variables. This study was supported by a Grant-in-Aid from the American Heart Association.
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