Editor's Note: This Article of the Month is based on Singh N, Van Craeyveld E, Tjwa M, et al. Circulating apoptotic endothelial cells and apoptotic endothelial microparticles independently predict the presence of cardiac allograft vasculopathy. Journal of the American College of Cardiology 2012; In press.

Article Summary

Background and Objective: Cardiac allograft vasculopathy (CAV) in heart transplant recipients is characteriszd by the coexistence of diffuse fibromuscular intimal hyperplasia and focal atherosclerosis.^1,2 In addition to malignancy, CAV is the most important cause of death in heart transplant recipients after the first year.^1,3,4

According to the response to injury concept of CAV, vascular lesions are the result of cumulative endothelial injury induced by alloimmune responses and non-alloimmune insults.^1,5 Following endothelial cell detachment induced by prolonged activation of endothelial cells or immunological injury, endothelial cells can be detected in the peripheral blood as viable circulating endothelial cells (CECs) or as apoptotic CECs.^6,7 Endothelial microparticles arising from exocytic budding following endothelial cell activation or following apoptosis constitute another biomarker of endothelial injury.^8-10 The process of endothelial injury is counteracted by endothelial repair mechanisms involving endothelial progenitor cells (EPC) and pro-angiogenic hematopoietic progenitor cells (HPCs).

In a cross-sectional study,¹¹ we investigated whether biomarkers related to endothelial injury and endothelial repair discriminate between CAV negative and CAV positive heart transplant recipients.

Methods: Fifty-two patients undergoing coronary angiography between 5 and 15 years after heart transplantation were recruited in this single-center study. Cell culture was used for quantification of circulating EPC number and HPC number and for analysis of EPC function. In addition, EPCs were analysed by flow cytometry. EPC concentration quantified by FACS was defined as the circulating number of CD34 VEGFR-2 (vascular endothelial growth factor receptor-2) double positive cells.^12,13

CECs were identified by FACS as CD45^- CD31^bright VEGFR-2⁺ mononuclear cells.¹⁴ Annexin V staining allows to distinguish viable and apoptotic CECs.¹⁵ Endothelial microparticles analysed by FACS were defined as CD144 (VE-Cadherin)⁺ CD42a^- microparticles.¹⁶ Annexin V binding was used to discriminate between apoptotic and non-apoptotic microparticles.

Results: EPC number and EPC function did not differ between CAV negative and CAV positive patients. In univariable models, age, creatinine, steroid dose, granulocyte colony-forming units, apoptotic CECs, and apoptotic endothelial microparticles discriminated between CAV positive and CAV negative patients. Both apoptotic CECs and apoptotic endothelial microparticles were independent predictors of CAV positive patients, and provided high discrimination (C statistic 0.812; 95% CI 0.692-0.932), as well as adding significant information (X²=18.4; df=2; p=0.0001). The C-statistic corresponding to this model was 0.855 (95% CI 0.756-0.953). When recipient age in these models was substituted by time after heart transplantation or age of the transplanted heart, results were essentially unaltered. Adding steroid dose to the model improved the C-statistic to 0.926 (95% CI 0.851-1.00). Finally, including apoptotic CECs together with endothelial apoptotic microparticles in the model with age, creatinine, and steroid dose added significant diagnostic value (X²=16.3; df=2; p=0.0003).

Conclusions: The concentration of apoptotic endothelial microparticles and of apoptotic CECs was significantly different between CAV positive and CAV negative patients (c-statistic of 0.812), and provided high discriminative ability between CAV positive and CAV negative patients.

Perspectives: In several logistic regression models, the introduction of apoptotic CECs and apoptotic endothelial microparticles consistently resulted in added value, indicating that these biomarkers are robust independent predictors. Whereas the final model in the current study was restricted to age, creatinine, apoptotic CECs, and apoptotic endothelial microparticles, the latter two parameters remain significant predictors. Importantly, the discriminative ability of these two biomarkers was also preserved in models in which recipient age was replaced by time after transplantation or by age of the transplanted heart.

The high discriminative ability of apoptotic CECs and apoptotic endothelial microparticles provides a solid foundation for the further development of clinical prediction models of CAV in the framework of prospective studies that evaluate CAV by coronary intravascular ultrasound. Prospective prediction models may lead to a more rational and more tailored use of coronary angiography and intravascular ultrasound to higher risk patients, reducing cost and patient risk. In addition, risk prediction models may allow timely interventions and assist in the design of new randomized clinical trials that can optimize therapy in heart transplant recipients.

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References

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Keywords: Allografts, Annexin A5, Antigens, CD, Apoptosis, Atherosclerosis, Coronary Angiography, Creatinine, Cross-Sectional Studies, Endothelial Cells, Heart Transplantation, Hematopoietic Stem Cells, Hyperplasia, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factor Receptor-2

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