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

https://doi.org/10.1016/j.ijcard.2014.06.062Get rights and content

Highlights

  • Cell culture, human heart, and blood were studied to assess links of CKD and CVD.

  • Myocardial fibrosis increases and capillary supply are decreased in CKD.

  • Endothelial to mesenchymal transition (EndMT) is increased in CKD.

  • Circulating inhibitors of angiogenesis are increased in CKD.

  • EndMT and angiogenesis inhibitors may cause fibrosis and capillary loss in CKD.

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 (Ptrend = 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.

Introduction

Individuals with chronic kidney disease (CKD) have high risks of developing and dying from cardiovascular (CV) disease and these risks are not fully explained by traditional risk factors [1]. Strong associations of novel factors with CV events, a failure of standard therapies to substantively impact mortality in advanced CKD, and an outsize risk of sudden death relative to myocardial infarction suggest that unique features underlie CVD in the setting of uremia [2].

Experimental models of uremia are characterized by myocardial fibrosis, loss of myocardial capillaries, and inhibition of ischemia driven neo-angiogenesis [3], [4]. These changes increase capillary to myocyte distance, alter oxygen delivery, and disrupt myocardial conduction, thereby facilitating propagation of arrhythmias. Experimental studies also demonstrate altered nitric oxide (NO) bioavailability in uremia [5] which may induce secondary changes in the activity and concentration of additional angiogenesis inhibitors [6], [7], [8] thereby contributing to the observed myocardial fibrosis and capillary rarefaction. However, similar data in human disease remains sparse. We undertook this study to characterize for the first time changes in human myocardial pathology across the spectrum of CKD and assess associations with NO-related circulating angiogenesis inhibitors and their effects on human coronary artery endothelial cells (HCAECs).

Section snippets

Autopsy cohort

Autopsies performed at Brigham & Women's Hospital (BWH) between 2004 and 2006 (n = 45) were included. Cases without sufficient data to estimate kidney function, history of acute kidney injury lasting > 1 week, cardiac transplant, active cancer, prior thoracic irradiation, treatment with anthracyclines, congenital heart disease, idiopathic or viral cardiomyopathy, or insufficient tissue were excluded. Medical history and laboratory data were extracted from clinical records. Kidney function was

Evaluation of cardiac fibrosis

The risk of sudden cardiac death increases dramatically as GFR declines [13]. Sudden cardiac death and overall mortality on the other hand are strongly associated with the presence of cardiac fibrosis [14]. We therefore aimed to systematically evaluate amount of cardiac fibrosis over various stages of CKD by analyzing heart tissue obtained from autopsy.

Among our autopsy cohort of 45 individuals, we identified 21 subjects with preserved kidney function, 17 with stage 3–4 CKD, and 7 with ESRD (

Discussion

We found that myocardial fibrosis and EndMT increased while microvascular supply decreased significantly with CKD severity. In addition, the concentration of circulating angiogenesis and NO inhibitors increased with CKD severity while serum from patients with more severe CKD inhibited proliferation and increased apoptosis of cultured coronary endothelial cells. Finally, ADMA, ANG, TSP and END had qualitatively similar effects on cultured endothelial cells as uremic serum.

CKD is strongly

Funding

This work was supported by funds of the University Medical Center of Göttingen (EMZ and MZ); the Paul Teschan Research Fund; the Carl Gottschalk Award of the American Society of Nephrology; the American Heart Association Scientist Development Grant [0735638N] (DMC), NIH RO1 HL070938 (JH), grants ZE523/3-1 and ZE523/2-1 by the Deutsche Forschungsgemeinschaft (MZ), the Cancer Prevention and Research Institute of Texas, the Metastasis Research Center at MD Anderson Cancer Center, and NIH grants

Conflict of interest

None declared. Author contributions—DMC and EMZ—study design, conduct of experiments, analysis of data, and drafting of manuscript. RP, RK, AMH—study design, conduct of experiments, and analysis of study. MZ and JH—analysis of data and drafting of manuscripts. AC, XX, XL—conduct of experiments and analysis of data. All authors approved the final draft.

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