Fig 1.
Endothelial TNAP activity in human myocardium.
(A) Fifteen samples were analyzed—five from non-failing donor hearts (#1–5); five from ischemic HF (#6–10), and five from idiopathic dilated HF patients (#11–15); serial sections from each sample were stained with oil red O (hematoxylin counterstain), AP activity, and alizarin red; microscopic fields containing arteries were identified and captured; arrows, small arteries. (B) Quantification of AP activity in each category of samples expressed as AP-positive area per high power field (HPF). (C) Representative consecutive sections from sample #5 (non-failing) stained for AP activity in the absence (left) or presence (right) of 12.5 mM of L-homoarginine, a specific TNAP inhibitor. (D) A photomerged overlay image of AP activity (white) and alizarin red staining (brown) from sample #15 (idiopathic dilated HF); (E) Top panels, AP activity (dark blue) in combination with α-smooth muscle actin (SMA) immunohistochemistry (brown); bottom panels, same vessels stained with SMA antibody (red) in combination with endothelial specific isolectin B4 (IB4, green); arrow, AP activity.
Fig 2.
A working hypothesis and the experimental model.
(A) Based on existing evidence from our animal model, we postulated that upregulated activity of endothelial TNAP can increase intimal calcification prior to development of atherosclerosis; “primary” calcified lesions in the intima can serve as focal points for lipid deposition or “secondary” atherosclerosis by increasing endothelial roughness. This type of accelerated lipid deposition might take place in coronary arteries of WHC-eTNAP mice that are rendered hypercholesterolemic by an atherogenic diet. Components of the model that are being tested in this study are highlighted in blue. (B) AP activity staining in the hearts of WHC and WHC-eTNAP mice and detection of vascular calcification in WHC-eTNAP mice at baseline (8 weeks of age) in coronary arteries prior to induction of hypercholesterolemia.
Table 1.
Blood chemistry of WHC and WHC-eTNAP mice (Mean ± SD).
Table 2.
Physiological characteristics of WHC and WHC-eTNAP mice (Mean ± SD).
Fig 3.
Atherosclerosis in the coronary arteries and the aortic root of WHC and WHC-eTNAP mice.
(A) Alizarin red staining for calcium, representative images. (B) Oil red O staining, hematoxylin counterstained; representative images. (C) Quantification of calcium (based on alizarin red staining) and lipids (oil red O staining) in the coronary arteries indicates that, under conditions of our model, calcification in the coronary arteries precedes and might be promoting lipid deposition. (D) Quantification of calcium (alizarin red staining) and lipids (oil red O staining) in the aortic root indicates that, under conditions of this experiment, calcification follows lipid deposition. All data were collected at baseline and at 16 weeks of age. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
Table 3.
Expression of osteogenic and chondrogenic markers in aortas of WHC and WHC-eTNAP mice at 16 weeks of age (arbitrary units; Mean ± SD).
Fig 4.
Coronary artery lesions in WHC-eTNAP mice.
(A) Consecutive sections of a left coronary artery (LCA) and a septal branch of the right coronary artery (RCA) from a 16-weeks-old WHC mouse with hypercholesterolemia (H&E staining). Both vessels are unaffected by atherosclerosis. (B) Consecutive sections of LCA and RCA from a 16-weeks-old WHC-eTNAP mouse with hypercholesterolemia (H&E staining). Both vessels are affected by atherosclerosis, with the RCA being severely stenotic. (C) Immunohistochemical detection of osteocalcin (green) in 16-weeks-old WHC and WHC-eTNAP mice with hypercholesterolemia. Sections were counterstain with DAPI (blue). (D) Sections containing the RCA from 13-weeks-old WHC and WHC-eTNAP mice with hypercholesterolemia were stained with picrosirius red for collagen. (E) Oil red O staining (red) of the LCA from 16-weeks-old WHC and WHC-eTNAP mice with hypercholesterolemia, counterstained with hematoxylin (purple).
Fig 5.
Effects of SBI-425 on plasma alkaline phosphatase activity, plasma pyrophosphate (PPi), and atherosclerosis in WHC-eTNAP mice.
(A) Plasma alkaline phosphatase activity was measured in plasma from non-fasted mice collected one to three weeks after initiation of the treatment protocol. (B) Plasma PPi was measured in plasma from non-fasted mice collected from a subset of animals one week after initiation of the treatment protocol. (C) Alizarin red staining for calcium is shown, representative images. (D) Oil red O staining, hematoxylin counterstained; representative images. (E) Quantification of calcium (based on alizarin red staining) and lipids (Oil red O staining) in coronary arteries. (F) Quantification of calcium (alizarin red staining) and lipids (Oil red O staining) in aortic roots. (C-F) Data were collected at 13 weeks of age; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Table 4.
Effect of SBI-425 on plasma calcium, phosphorus, and lipids (Mean ± SD).
Table 5.
Physiologic parameters of WHC and WHC-eTNAP mice in the SBI-425 study (Mean ± SD).