Regulation of vascular smooth muscle cell calcification by extracellular pyrophosphate homeostasis: synergistic modulation by cyclic AMP and hyperphosphatemia

Abstract

Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate (PP(i)), and regulated generation and homeostasis of extracellular PP(i) is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular PP(i) generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular PP(i) mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate (P(i)) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular PP(i). Attenuated PP(i) accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades PP(i). Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular P(i) alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated P(i) induced decreases in the ATP release pathway(s) that supports ENPP1 activity; this resulted in markedly reduced rates of PP(i) accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) alpha-actin, SM22alpha, and calponin. Notably, addition of exogenous ATP (or PP(i) per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular PP(i) generation mediated by both ANK- and ATP release-dependent mechanisms serves as a critical regulator of VSMC calcification.

Fig. 1.

Potentiation of inorganic phosphate (P_i)-induced vascular smooth muscle cell (VSMC) matrix mineralization by cAMP signaling: time course and dependence on extracellular P_i concentration. A: quantification of Alizarin Red S staining after treatment of VSMC for the indicated days in culture in the absence or presence of 1 μM forskolin (FSK), 5 mM P_i, or both 1 μM FSK + 5 mM P_i. Abs₅₇₀, absorbance at 570 nm. Inset: qualification of Alizarin Red S stain in VSMC treated for 10 days with the indicated stimuli. B: matrix mineralization time course of VSMC treated for the indicated days in culture in the absence or presence of 5 mM P_i + 1 μM FSK. C: P_i dose response (3 or 5 mM) in VSMC treated for 10 or 12 days in the absence or presence of 1 μM FSK. For all experiments, fresh growth medium supplemented with FSK, P_i, or FSK + P_i was added every 3 days. Data represent means ± SE; n ≥ 3. *P < 0.05 vs. control.

Fig. 2.

VSMC matrix mineralization is correlated with decreased accumulation of extracellular inorganic pyrophosphate (PP_i) and altered expression of PP_i homeostatic gene products. A: Western blot of VSMC cell extracts probed for SMC marker proteins α-SM-actin, SM22α, and calponin for the indicated days in culture in the absence or presence of 1 μM FSK, 5 mM P_i, or FSK + P_i with β-actin and GAPDH as loading controls. Signal intensities of the GAPDH bands in each lane were quantified by chemifluorescence imaging and normalized relative to the GAPDH band intensity of control VSMC (1st lane). B: quantitative PCR (qPCR) analysis of PP_i homeostatic proteins tissue nonselective alkaline phosphatase (TNAP), ANK, and ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1), along with the osteochondrogenic marker Runx2 in VSMC treated for the indicated days in culture in the absence and presence of 1 μM FSK, 5 mM P_i, or FSK + P_i. C: alkaline phosphatase activity, measured by the rate of para-nitrophenylphosphate (pNPP) hydrolysis, in lysates of VSMC treated for the indicated days in culture in the absence or presence of 1 μM FSK, 5 mM P_i, or FSK + P_i. D: extracellular levels of PP_i generation at the 2 h time point after transfer of VSMC to basal saline assay medium that were treated for the indicated days in culture in the absence or presence of 1 μM FSK, 5 mM P_i, or FSK + P_i. Data represent means ± SE; n ≥ 3. *P < 0.05 vs. control.

Fig. 3.

cAMP signaling decreases the rate of extracellular PP_i accumulation by VSMC cultured with control or elevated levels of extracellular P_i. Time course of extracellular levels of PP_i generation following transfer of VSMC to basal saline assay medium that were treated for 1 day in the absence (Con) or presence of 5 mM P_i or 5 mM P_i + FSK (A); 10 days in the absence (Con) or presence of 3 mM P_i, 1 μM FSK, or 3 mM P_i + FSK (B); or 10 days in the absence (Con) or presence of 5 mM P_i, 1 μM FSK, or 5 mM P_i + FSK (C). Data represent means ± SE; n ≥ 3. *P < 0.05 vs. control.

Fig. 4.

cAMP-induced inhibition of autocrine PP_i accumulation in VSMC: pharmacology, kinetics, and reversibility. A: extracellular levels of PP_i generation after transfer of VSMC to basal saline assay medium that were treated for 24 h in the absence (Con) or presence of 1 μM FSK. B: time course of VSMC treated with 1 μM FSK; data represent the extracellular levels of PP_i generation at the 2 h time point after transfer of VSMC to basal saline assay medium. C: FSK dose response of VSMC treated for 24 h; data represent the extracellular levels of PP_i generation at the 2 h time point after transfer of VSMC to basal saline assay medium. D: extracellular levels of PP_i generation at the 2 h time point after transfer of VSMC to basal saline assay medium that were treated in the absence (Con) or presence of 1 μM FSK for 1 day (FSK-1d), 2 days (FSK-2d), or 1 day followed by the removal of FSK and incubated for an additional day (FSK-1d, no FSK-1d). E: extracellular levels of PP_i generation as % of control at the 2 h time point after transfer of VSMC to basal saline assay medium that were treated for 24 h in the absence (Con) or presence of 1 μM FSK, 200 μM 3-isobutyl-1-methylxanthine (IBMX), or 100 μM 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate (CPT-cAMP). F: extracellular levels of PP_i generation at the 2 h time point in the absence or presence of 5 mM levamisole (Lev) after transfer of VSMC to basal saline assay medium that were treated in the absence (Con) or presence of 1 μM FSK for 24 h. Data represent means ± SE; n ≥ 3. *P < 0.05 vs. control.

Fig. 5.

ENPP1 ectonucleotidase activity is maintained at control levels in VSMC stimulated by cAMP or elevated extracellular P_i. A: ENPP1 Western blot of VSMC cell extracts treated for the indicated days in culture in the absence or presence of 1 μM FSK, 5 mM P_i, or FSK + P_i. HEK-293 cells stably expressing green fluorescent protein (GFP) or ENPP1 were used as controls (last 2 lanes on right). B: representative HPLC chromatogram of extracellular medium from intact control VSMC pulsed with 300 μM exogenous β,γ-methylene ATP (MeATP) and incubated for the indicated times. Ado, adenosine. Inset: cumulative time course data of MeATP (300 μM) hydrolysis in control VSMC based on calibrated peak heights detected by absorbance from the HPLC chromatograms; control half-life (t_1/2) = 12.425 ± 0.19 min. Table shows t_1/2 of 300 μM MeATP hydrolysis based on HPLC analysis of VSMC treated for the indicated days (d) in culture in the presence of 1 μM FSK, 5 mM P_i, or P_i + FSK. C and D: extracellular levels of PP_i after transfer of VSMC to basal saline assay medium incubated in the absence (autocrine) or presence of 500 nM ATP after treatment of VSMC for 24 h in the absence (C) or presence (D) of 1 μM FSK. E: extracellular levels of ATP after transfer to basal saline assay medium incubated in the presence of 500 nM ATP after treatment of VSMC in the absence (control) or presence of 1 μM FSK for 24 h. Data represent the range ± SD from a representative experiment repeated 3 times.

Fig. 6.

Autocrine ATP release is decreased in VSMC costimulated by cAMP and elevated extracellular P_i. A: extracellular levels of ATP in the absence or presence of 300 μM MeATP in VSMC treated for 1 or 10 days in culture in the absence or presence of 1 μM FSK, 5 mM P_i, or FSK + P_i. B: cumulative data of extracellular ATP levels taken at the 10 min time point after transfer to basal saline assay medium in the presence of 300 μM MeATP of VSMC treated for the indicated days in culture in the absence or presence of FSK, P_i, or FSK + P_i. Data represent means ± SE; n ≥ 3. *P < 0.05 vs. control.

Fig. 7.

Matrix mineralization of VSMC costimulated by cAMP and elevated extracellular P_i is suppressed by exogenous ATP. Quantification of Alizarin Red S staining after treatment of VSMC for 10 days in culture in the absence or presence of 1 μM FSK and 5 mM P_i and in the absence or presence of exogenous PP_i, ATP, CTP, or Ado (each at 50 μM). Fresh growth medium supplemented with FSK + P_i, exogenous PP_i, or nucleotides was added every 3 days. Data represent means ± SE; n = 3. *P < 0.05 vs. control; ^#P < 0.05 vs. 10-day FSK + P_i.