Chronic mineral dysregulation promotes vascular smooth muscle cell adaptation and extracellular matrix calcification

Abstract

In chronic kidney disease (CKD) vascular calcification occurs in response to deranged calcium and phosphate metabolism and is characterized by vascular smooth muscle cell (VSMC) damage and attrition. To gain mechanistic insights into how calcium and phosphate mediate calcification, we used an ex vivo model of human vessel culture. Vessel rings from healthy control subjects did not accumulate calcium with long-term exposure to elevated calcium and/or phosphate. In contrast, vessel rings from patients with CKD accumulated calcium; calcium induced calcification more potently than phosphate (at equivalent calcium-phosphate product). Elevated phosphate increased alkaline phosphatase activity in CKD vessels, but inhibition of alkaline phosphatase with levamisole did not block calcification. Instead, calcification in CKD vessels most strongly associated with VSMC death resulting from calcium- and phosphate-induced apoptosis; treatment with a pan-caspase inhibitor ZVAD ameliorated calcification. Calcification in CKD vessels was also associated with increased deposition of VSMC-derived vesicles. Electron microscopy confirmed increased deposition of vesicles containing crystalline calcium and phosphate in the extracellular matrix of dialysis vessel rings. In contrast, vesicle deposition and calcification did not occur in normal vessel rings, but we observed extensive intracellular mitochondrial damage. Taken together, these data provide evidence that VSMCs undergo adaptive changes, including vesicle release, in response to dysregulated mineral metabolism. These adaptations may initially promote survival but ultimately culminate in VSMC apoptosis and overt calcification, especially with continued exposure to elevated calcium.

Figure 1.

Dialysis vessels show time-dependent Ca accumulation in vitro. The Ca load in normal, predialysis, and dialysis vessel rings was quantified after incubation for 7, 14, and 21 d. Normal vessels did not increase Ca loading, whereas dialysis vessels showed maximal Ca load in response to Ca + P. (A) Incubation in high P medium (2.0 mM P + 1.8 mM Ca). The Ca load in normal vessels was 10.0 ± 2.4 versus 8.4 ± 3.6 versus 11.4 ± 1.6 μg/μl (P = 0.40), in predialysis vessels was 20.2 ± 5.8 versus 30.1 ± 11.9 versus 41.7 ± 7.2 μg/μl (P = 0.07), and in dialysis vessels was 176.3 ± 17.9 versus 298.2 ± 93.7 versus 726.7 ± 103.9 μg/μl (P < 0.0001) at 7, 14, and 21 d, respectively. (B) Incubation in high Ca + P medium (2.0 mM P + 2.7 mM Ca). The Ca load in normal vessels was 11.2 ± 1.9 versus 12.9 ± 3.5 versus 17.0 ± 6.0 μg/μl (P = 0.16), in predialysis vessels was 43.2 ± 13.8 versus 115.1 ± 49.0 versus 170.8 ± 35.5 μg/μl (P = 0.01), and in dialysis vessels was 325.7 ± 137.0 versus 711.0 ± 206.0 versus 1857.0 ± 90.0 μg/μl (P = 0.0007) at 7, 14, and 21 d, respectively. (C) Von Kossa staining of vessel rings after 14 d in calcifying media. Normal and predialysis vessels did not show any calcification in high P medium. In high Ca + P medium, some normal and predialysis vessels showed punctate calcification along the internal elastic lamina (inset). Dialysis vessels developed calcification in all in vitro conditions with large confluent areas of calcification in the high Ca + P medium (inset). M, medium; Ad, adventitia

Figure 2.

Ca is a more potent inducer of calcification than P. The Ca load in all vessel types was quantified after exposure to calcifying media with the same Ca × P of 5.4 mM² (3.0 mM P + 1.8 mM Ca and 2.0 mM P + 2.7 mM Ca) for 14 d. (A) The Ca load in normal vessels was unaffected (6.7 ± 2.4 versus 8.4 ± 3.6 versus 11.8 ± 2.9 versus 12.9 ± 3.5 μg/μl; P = 0.3), was increased in predialysis (19.7 ± 1.1 versus 30.1 ± 11.9 versus 47.9 ± 8.3 versus 115.1 ± 49.0 μg/μl; P = 0.03), and was maximum in dialysis vessels (129.0 ± 53.0 versus 298.2 ± 93.7 versus 350.0 ± 147.0 versus 711.0 ± 206.0 μg/μl; P < 0.0001) in response to in 1.0 mM P + 1.8 mM Ca, 2.0 mM P + 1.8 mM Ca, 3.0 mM P + 1.8 mM Ca, and 2.0 mM P + 2.7 mM Ca media, respectively. On comparing media with similar Ca × P, the Ca load was significantly increased in predialysis (P = 0.04) and dialysis (P = 0.02) vessels but not in normal vessels (P = 0.77). (B) Dialysis vessels with baseline von Kossa positivity (n = 6) showed greater calcification than baseline von Kossa–negative (n = 18) vessels after culture for 14 d in calcifying media. Ca load in von Kossa–positive vessels was 44.2 ± 5.1, 370.0 ± 25.1, and 1254.0 ± 406.2 μg/μl (P = 0.0005, ANOVA) versus 30.5 ± 5.4, 208.0 ± 78.6, and 476.3 ± 143.7 μg/μl (P < 0.001, ANOVA) in von Kossa–negative vessels in 1.0 mM P + 1.8 mM Ca, 2.0 mM P + 1.8 mM Ca, and 2.0 mM P + 2.7 mM Ca media, respectively. (C) Predialysis (n = 4) and dialysis (n = 6) vessels with similar baseline Ca loads after culture for 14 d in calcifying media showed increased Ca loading in dialysis vessels (31.5 ± 2.4 and 278.2 ± 105.0 μg/μl; P = 0.0008) compared with predialysis vessels (27.0 ± 3.3 and 48.7 ± 13.2 versus 96.8 ± 14.4 μg/μl; P = 0.03) in identical calcifying conditions. All probability values calculated by ANOVA.

Figure 3.

Osteogenic conversion of VSMCs in predialysis and dialysis vessels. (A) ALK levels in normal vessels were 5.7 (2.3 to 7.5) versus 4.2 IU/μl (3.2 to 4.8 IU/μl; P = 0.4), in predialysis vessels were 3.9 (2.4 to 15.1) versus 14.4 IU/μl (4.2 to 27.1 IU/μl; P = 0.03, and in dialysis vessels were 5.0 (5.7 to 19.3) versus 30.5 IU/μl (16.0 to 42.1 IU/μl; P = 0.001) after incubation in 1.0 mM P + 1.8 mM Ca and 2.0 mM P + 1.8 mM Ca media for 14 d, respectively. In 2.0 mM P + 2.7 mM Ca medium, ALK levels were significantly reduced in all vessel types (2.1 [0.9 to 3.1], 2.2 [1.0 to 2.9], and 5.7 IU/μl [3.8 to 8.9 IU/μl] in normal, predialysis, and dialysis vessels, respectively). (B) Dialysis vessels incubated in 2.0 mM P + 1.8 mM Ca medium with the addition of levamisole showed a decrease in ALK levels (31.1 [16.3 to 42.3] versus 17.1 [6.2 to 19.3]; P = 0.03). (C) Ca load in vessel rings from the aforementioned experiment remained unchanged (387.2 ± 22.0 versus 356.0 ± 37.0 μg/μl; P = 0.62). (D) Immunohistochemistry for Runx2 was performed in dialysis vessels after culture for 14 d in calcifying media. Runx2 positivity increased in high P medium and was significantly greater in high Ca + P medium. Note the cytoplasmic localization of Runx2 in dialysis vessels potentially reflecting alternative isoform usage in these vessels. (E) The number of Runx2-positive areas per unit area of tunica media was counted in dialysis vessels after incubation for 14 d. Runx2-positive areas were 16.6 ± 4.1, 24.9 ± 7.8, and 61.5 ± 10.9% (P = 0.002) in 1.0 mM P + 1.8 mM Ca, 2.0 mM P + 1.8 mM Ca, and 2.0 mM P + 2.7 mM Ca media, respectively.

Figure 4.

Dialysis vessels undergo VSMC loss as a result of apoptotic cell death. VSMC nuclei per unit area of tunica media were counted on hematoxylin-eosin–stained samples in all vessel types after in vitro culture for 14 d in high P and high Ca + P. (A) The VSMC number in normal vessels was 122.0 ± 3.9 versus 120.0 ± 5.8 versus 115.0 ± 4.7 cells per unit area (P = 0.09), in predialysis vessels was 118.0 ± 9.1 versus 118.0 ± 9.8 versus 108.0 ± 7.7 cells per unit area (P = 0.047) and in dialysis vessels was 85.0 ± 17.9 versus 82.0 ± 14.6 versus 59.0 ± 10.7 cells per unit area (P = 0.03) in 1.0 mM P + 1.8 mM Ca, 2.0 mM P + 1.8 mM Ca, and 2.0 mM P + 2.7 mM Ca media, respectively. (B) At similar baseline Ca loads, predialysis vessels had 120.5 ± 8.4 versus 114.3 ± 11.3 versus 107.0 ± 8.6 cells per unit area (P = 0.16), and dialysis vessels had 87.8 ± 9.1 versus 76.0 ± 9.9 versus 54.0 ± 6.7 cells per unit area (P = 0.003) in 1.0 mM P + 1.8 mM Ca, 2.0 mM P + 1.8 mM Ca, and 2.0 mM P + 2.7 mM Ca media, respectively. (C) Staining for α-smooth muscle actin (α-SM actin) was patchy in dialysis compared with predialysis and normal vessels; arrows show cystic areas denoting VSMC loss. TUNEL-positive areas were observed in dialysis vessels only, and the frequency of nuclear fragments was also increased. Inset shows hematoxylin-eosin staining of nuclear fragments (arrow) associated with a VSMC cyst in a dialysis vessel treated with Ca + P medium. (D) The number of TUNEL-positive cells per unit area of each vessel was counted and expressed as a percentage of total cell number after 14 d of incubation in calcifying media. Normal and predialysis vessels had <0.5% apoptotic cells in all in vitro conditions, whereas dialysis vessels showed a significant increase (2.6 [0.0 to 6.2], 2.9 [0.0 to 8.6], and 7.7% [0.0 to 37.9%] TUNEL-positive cells (P = 0.03) in 1.0 mM P + 1.8 mM Ca, 2.0 mM P + 1.8 mM Ca, and 2.0 mM P + 2.7 mM Ca media, respectively). (E) On addition of ZVAD to Ca + P–treated dialysis vessels, the Ca load reduced from 363.7 ± 28.0 versus 278.0 ± 34.0 μg/μl (P = 0.04) but remained unchanged in vessels treated in high P medium alone (279.2 ± 24.1 versus 265.3 ± 30.3 μg/μl; P = 0.12). All probability values were calculated by ANOVA.

Figure 5.

Immunohistochemistry for vesicle markers and EM analysis of vessels. (A) Immunohistochemistry for the vesicle marker annexin VI showed minimal deposition in normal vessels after exposure to calcifying media for 14 d but increased deposition in dialysis vessels. (B) A similar pattern was observed for the vesicle-associated protein fetuin-A, which was absent in normal vessels but heavily deposited in dialysis vessels. Fetuin is present intracellularly as well as deposited in the VSMC matrix. All panels show α-SM actin co-staining (blue) and fetuin-A (brown). (C, i) EM showing calcified mitochondria (arrow) in VSMCs from control vessels exposed to high Ca + P medium in vitro. *Degenerate VSMC with remnant calcified mitochondria and membrane debris. (ii) Enlargement of calcified mitochondria. (iii) Enlargement of rare membrane debris found associated with degenerate VSMCs in control vessels. This was never calcified, and no evidence of ECM calcification was observed in normal vessels. (iv) Crystalline ECM calcification (arrow) was observed in calcified dialysis vessels after exposure to Ca + P in vitro. These VSMCs exhibited intact mitochondria (arrowheads). (v) Vesicle deposition in the ECM of dialysis vessels exposed to Ca + P occurred in close proximity to healthy intact VSMCs (membranes arrow) and associated with elastin. (vi) Enlargement of vesicles. Note that a large number of these vesicles show crystalline apatite within the lumen (arrow). Bar = 0.5 μm.

Figure 6.

Model shows VSMC phenotypic adaptation in response to mineral dysregulation in CKD. VSMCs that fail to adapt to a synthetic phenotype and release vesicles to protect against Ca overload will eventually undergo necrosis. In contrast, VSMCs that release vesicles do not succumb to intracellular Ca overload but deposit Ca in the ECM, which eventually calcifies. This process eventually results in apoptosis of VSMCs.