Angiopoietin-2-induced arterial stiffness in CKD

Abstract

The mechanism of vascular calcification in CKD is not understood fully, but may involve collagen deposition in the arterial wall upon osteo/chondrocytic transformation of vascular smooth muscle cells (VSMCs). Increased levels of circulating angiopoietin-2 correlate with markers of CKD progression and angiopoietin-2 regulate inflammatory responses, including intercellular and vascular adhesion and recruitment of VSMCs. Here, we investigate the potential role of angiopoietin-2 in the pathogenesis of arterial stiffness associated with CKD. In a cohort of 416 patients with CKD, the plasma level of angiopoietin-2 correlated independently with the severity of arterial stiffness assessed by pulse wave velocity. In mice subjected to 5/6 subtotal nephrectomy or unilateral ureteral obstruction, plasma levels of angiopoietin-2 also increased. Angiopoietin-2 expression markedly increased in tubular epithelial cells of fibrotic kidneys but decreased in other tissues, including aorta and lung, after 5/6 subtotal nephrectomy. Expression of collagen and profibrotic genes in aortic VSMCs increased in mice after 5/6 subtotal nephrectomy and in mice producing human angiopoietin-2. Angiopoietin-2 stimulated endothelial expression of chemokines and adhesion molecules for monocytes, increased Ly6C(low) macrophages in aorta, and increased the expression of the profibrotic cytokine TGF-β1 in aortic endothelial cells and Ly6C(low) macrophages. Angiopoietin-2 blockade attenuated expression of monocyte chemokines, profibrotic cytokines, and collagen in aorta of mice after 5/6 subtotal nephrectomy. This study identifies angiopoietin-2 as a link between kidney fibrosis and arterial stiffness. Targeting angiopoietin-2 to attenuate inflammation and collagen expression may provide a novel therapy for cardiovascular disease in CKD.

Figure 1.

Plasma levels of Angpt2 increase and positively correlate with PWV in patients with CKD. (A–D) Univariate regression analysis shows the linear correlation of eGFR with plasma levels of Angpt1 (A), Angpt2 (B), VEGF-A (C), and sTie-2 (D). (E) Univariate regression analysis shows the linear correlation of PWV with plasma levels of Angpt2 in patients with CKD. The plasma levels of Angpt1, Angpt2, VEGF-A, and sTie-2 are expressed as the natural logarithm (ln).

Figure 2.

Angpt2 is increased in plasma and kidney after 5/6Nx. (A) Plasma levels of Angpt1 and Angpt2 and the ratio of plasma Angpt2/Angpt1 after 5/6Nx. (B) qPCR for transcripts of Angpt1 and Angpt2 in different organs 8 weeks after 5/6Nx. (C) qPCR time course of kidney for transcripts of Angpt1 and Angpt2. Expression levels are normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). n=10 per time point. *P<0.05; ^†P<0.01 versus sham at each time point.

Figure 3.

Angpt2 is increased in injured renal tubular epithelial cells after 5/6Nx. (A) Confocal microscopic images showing Angpt2 expression in injured renal tubular epithelial cells and glomeruli after 5/6Nx. (B) In situ hybridization showing increased transcripts of Angpt2 in injured renal tubular epithelial cells, glomeruli, and interstitial cells after 5/6Nx. The specificity of in situ hybridization is confirmed using a sense RNA probe. T, renal tubular epithelial cell; G, glomeruli; LTL, L. tetragonolobus lectin; Kim-1, kidney injury molecule-1. Scale bar, 20 μm in A; 25 μm in B.

Figure 4.

Increased transcripts of profibrotic and proinflammatory genes in aorta of mice after 5/6Nx. (A–D) qPCR for transcripts of COL1A1 and COL3A1 (A), TGFB1 and PDGF-B (B), CCL2, CCR2, CX3CL1, and CX3CR1 (C), and ICAM1 and VCAMA1 (D) in aortas of mice after 5/6Nx. Expression levels are normalized by glyceraldehyde 3-phosphate dehydrogenase. n=15 per group. *P<0.05; ^†P<0.01; ^‡P<0.001.

Figure 5.

Aortic VSMCs generate collagen transcripts in mice producing human Angpt2. (A) Plasma levels of human Angpt2 in Coll-GFP^Tg mice administered with AdAngpt2 and AdCon. (B and C) Immunofluorescence images showing that α-SMA⁺ aortic VSMCs generate COL1A1 transcripts, as indicated by increased Coll-GFP, in mice with AdAngpt2. Coll-GFP expression in adventitial fibroblasts does not change (arrows in B). C57BL/6 wild-type mice serve as the negative control for GFP. Autofluorescence from elastic fibers is indicated (arrowheads in C). (D) qPCR showing increased transcripts of COL1A1 and COL3A1 in aorta of mice with AdAngpt2. (E) qPCR showing increased transcripts of COL1A1, CX3CL1, and TGFB1 in primary cultured VSMCs stimulated by recombinant TGF-β1 (5 ng/ml), but not by Angpt2 (500 ng/ml), for 24 hours. Angpt2 of different concentrations shows the same results. Expression levels are normalized by glyceraldehyde 3-phosphate dehydrogenase. ^†P<0.01; ^‡P<0.001. αSMA, α-smooth muscle actin. Scale bar, 20 μm in C.

Figure 6.

Angpt2 induces discrete subpopulation of macrophages defined by Ly6C in aorta. (A) Immunofluorescence images showing that Tie-2 receptor is mainly expressed in CD31⁺ endothelial cells of the aorta from normal control mice. (B) FACS analysis showing that the Tie-2 receptor is also expressed in CD11b⁺ macrophages of the aorta from normal control mice. (C) qPCR showing increased transcripts of chemokine and chemokine receptors in aorta of mice with AdAngpt2. (D and E) FACS analysis showing that Ly6C^low subpopulation of CD11b⁺ macrophages is induced in aorta of mice with AdAngpt2. (F) qPCR of M1- and M2-biased cytokines of sorted CD11b⁺ macrophages defined by Ly6C expression. Expression levels are normalized by glyceraldehyde 3-phosphate dehydrogenase. *P<0.05; ^†P<0.01; ^‡P<0.001. Scale bar, 20 μm in A.

Figure 7.

Angpt2 induces proinflammatory chemokines and adhesion molecules in endothelial cells. (A) qPCR showing increased transcripts of CCL2, CX3CL1, ICAM1, and TGFB1 in FACS sorted aortic endothelial cells from mice with AdAngpt2. (B) qPCR for the transcripts of chemokines, adhesion molecules, and profibrotic cytokines in primary cultured aortic endothelial cells stimulated with recombinant Angpt2 (500 ng/ml) for 24 hours. Expression levels are normalized by glyceraldehyde 3-phosphate dehydrogenase. ^†P<0.01; ^‡P<0.001.

Figure 8.

Blocking Angpt2 with recombinant protein L1-10 attenuates the transcripts of profibrotic and proinflammatory genes in aorta of mice after 5/6Nx. (A–D) qPCR for the transcripts of TGFB1 (A), COL1A1 and COL3A1 (B), CCL2, CCR2, CX3CL1, and CX3CR1 (C), and ICAM1 and VCAM1 (D) in aortas of sham, 5/6Nx receiving sFc or L1-10. Expression levels are normalized by glyceraldehyde 3-phosphate dehydrogenase. n=15 per group. *P<0.05; ^†P<0.01; ^‡P<0.001.