Increased concentration of circulating angiogenesis and nitric oxide inhibitors induces endothelial to mesenchymal transition and myocardial fibrosis in patients with chronic kidney disease - PubMed (original) (raw)

Increased concentration of circulating angiogenesis and nitric oxide inhibitors induces endothelial to mesenchymal transition and myocardial fibrosis in patients with chronic kidney disease

David M Charytan et al. Int J Cardiol. 2014 Sep.

Abstract

Background: Sudden cardiovascular death is increased in chronic kidney disease (CKD). Experimental CKD models suggest that angiogenesis and nitric oxide (NO) inhibitors induce myocardial fibrosis and microvascular dropout thereby facilitating arrhythmogenesis. We undertook this study to characterize associations of CKD with human myocardial pathology, NO-related circulating angiogenesis inhibitors, and endothelial cell behavior.

Methods: We compared heart (n=54) and serum (n=162) samples from individuals with and without CKD, and assessed effects of serum on human coronary artery endothelial cells (HCAECs) in vitro. Left ventricular fibrosis and capillary density were quantified in post-mortem samples. Endothelial to mesenchymal transition (EndMT) was assessed by immunostaining of post-mortem samples and RNA expression in heart tissue obtained during cardiac surgery. Circulating asymmetric dimethylarginine (ADMA), endostatin (END), angiopoietin-2 (ANG), and thrombospondin-2 (TSP) were measured, and the effect of these factors and of subject serum on proliferation, apoptosis, and EndMT of HCAEC was analyzed.

Results: Cardiac fibrosis increased 12% and 77% in stage 3-4 CKD and ESRD and microvascular density decreased 12% and 16% vs. preserved renal function. EndMT-derived fibroblast proportion was 17% higher in stage 3-4 CKD and ESRD (P trend = 0.02). ADMA, ANG, TSP, and END concentrations increased in CKD. Both individual factors and CKD serum increased HCAEC apoptosis (P=0.02), decreased proliferation (P=0.03), and induced EndMT.

Conclusions: CKD is associated with an increase in circulating angiogenesis and NO inhibitors, which impact proliferation and apoptosis of cardiac endothelial cells and promote EndMT, leading to cardiac fibrosis and capillary rarefaction. These processes may play key roles in CKD-associated CV disease.

Keywords: Angiogenesis inhibitor; CKD; Cardiovascular disease; ESRD; Endothelial to mesenchymal transition; Fibrosis.

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Figures

Figure 1. Evaluation of fibrosis and microvessels in the LV

(A) Pictures show representative left ventricular sections stained with Masson-Trichrome to visualize fibrotic tissue (stained in blue) (B) pictures shows immunohistochemical anti-CD31 staining to label microvessels (stained in brown) (A, B) left images shows heart tissue from an individual with preserved GFR, middle images from a patient with stage 3-4 CKD, and right images from a dialysis patient. (C-F) Quantitative analysis of Masson-Trichrome and anti-CD31 stained sections from n=21 preserved GFR, n=17 stage 3-4 CKD, and n=7 dialysis patients for (C) Fibrosis, (D) Microvascular density, (E) Myocyte size, and (F) Myocyte density.

Figure 2. EndMT in the heart: Confocal microscopy and qPCR expression analysis

(A) Representative confocal images of left ventricular tissue after immunofluorescent staining with antibodies for the fibroblast marker FSP1 (green) and endothelial cell marker CD31 (red). Nuclei are stained with DAPI (blue). Top row shows tissue from an individual with preserved renal function, middle row from a patient with stage 3-4 CKD, and the bottom row from a dialysis patient. Dual FSP1/CD31 positive cells, indicative of EndMT (arrows), increase with decreasing renal function. Magnification x63. (B-D) Quantitative analysis of n=11 preserved GFR, n=8 stage 3-4 CKD, and n=4 dialysis patients for (B) FSP+ cells/visual field, (C) FSP1/CD-31 double positive cells/visual field, indicative of cells undergoing EndMT. (D) Ratio of FSP1/CD31 double positive cells to all FSP+ cells, indicative of the fraction of EndMT-derived FSP1-positive fibroblasts. (E, F, G) Bar graphs show quantitative real time PCR for EndMT marker genes SLUG (E), SNAIL (F), and TWIST (G) in right atrial appendage of individuals with preserved function (n=3), with CKD stage 3-4 (n=3), and dialysis patients (n=3).

Figure 3. Effect of CKD serum on human coronary artery endothelial cells (HCAECs): proliferation, apoptosis, and EndMT

(A, B) Representative light microscopy images of HCAEC after 6 days of incubation with serum from subjects with preserved renal function (A) and with CKD (B). Medium with serum was changed every other day. Number of cells decreased upon CKD serum incubation and cell morphology of surviving cells changed towards more spindle shaped as compared to incubation with serum from an individual with preserved renal function. (C, D) HCAEC were incubated with serum from individuals with preserved renal function (n=30), or from CKD patients stage 2 (n=58), stage 3-4 (n=60), or stage 5 (n=14), and relative apoptosis (as assessed by the Caspase-Glo 3/7 assay) and proliferation (as assessed by the WST-1 assay) were measured. (C) Relative proliferation of HCAECs decreased with more severe CKD. (D) Relative apoptosis of HCAEC increased with increasing CKD severity. (E-G) Bar graphs show quantitative real time PCR for EndMT marker genes SLUG (E), SNAIL (F), and TWIST (G) (all relative to GAPDH) in HCAEC upon incubation with healthy (n=3) versus CKD (n=9, 3 per stage) serum. Both TWIST and SNAIL are increased upon incubation with serum from CKD stages 3 through 5.

Figure 4. Serum concentrations of anti-angiogenic factors in CKD patients

Box plots show serum levels of (A) the NO-synthase inhibitor ADMA and (B-D) the angiogenesis inhibitors (B) Endostatin, (C) Thrombospondin-2 and (D) Angiopoietin-2. Boxes span the minimum and maximum values (log transformed). Horizontal lines represent the median. Vertical lines above and below each box encompass maximal and minimal values. Serum of individuals with preserved renal function (n= 30), or from CKD patients stage 2 (n=58), stage 3-4 (n=60), or stage 5 (n=14) was used for all of these measurements.

Figure 5. Effect of recombinant Angiopoietin-2, Thrombospondin-2, ADMA and Endostatin on proliferation, apoptosis and EndMT of HCAECs

HCAECs were treated with different concentrations of recombinant angiopoietin-2 (A-C), thrombospondin-2 (D-F), ADMA (G-I), or endostatin (J-L). Bar graphs show the effect of these factors on HCAEC proliferation (A, D, G, J), HCAEC apoptosis (B, E, H, K), and EndMT marker expression SLUG and TWIST (C, F, I, L).

Figure 6. Schematic for mechanisms of fibrosis and capillary rarefaction in CKD

High circulating concentrations of the angiogenesis inhibitors asymmetric dimethylarginine (ADMA), endostatin (END), angiopoietin-2 (ANG), and thrombospondin-2 (TSP) in CKD lead to EndMT causing microvascular rarefaction, fibroblast accumulation, and cardiac fibrosis.

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Increased concentration of circulating angiogenesis and nitric oxide inhibitors induces endothelial to mesenchymal transition and myocardial fibrosis in patients with chronic kidney disease - PubMed (original) (raw)