Endomyocardial biopsy (EMB) is currently the gold standard method for detection and confirmation of the allograft rejection. Several methods have been proposed for an accurate diagnosis, yet none so far has proven to be adequate or specific enough to replace the status quo [59]. The necessity to replace endomyocardial biopsy is to avoid an invasive method and to provide the clinician with a tool with fast and accurate results. In current practice, major adjunct methods are used for monitoring rejection, such as electrocardiography, electrophysiology, radio-isotopic techniques, cyto-immunologic monitoring, and magnetic resonance imaging [60, 61]. Among the proposed biochemical markers are urinary polyamines [61], neopterins [62], prolactin [63], ­beta-2-­microglobulins [64], and brain natriuretic peptide (BNP) [65], none of which had fulfilled the need for sensitivity.

New high-throughput analyses that utilize protein or gene expression analysis techniques, cell-mediated immune assays, and fluorescence-activated cell sorter analysis of cellular subpopulations have the potential to provide the clinicians with more accurate markers of allograft rejection. Advancements in microarray technologies and bioinformatics allow ­researchers to determine specific proteins, genes, metabolites and pathways that are involved in normal and dysfunctional processes [66]. These major methodological developments create the possibility for discovery of new markers, while also presenting novel targets for therapy. Human biopsy materials obtained from ailing tissue are being used to investigate the etiology of diseases; a good example would be cardiac biopsies used for tetralogy of Fallot [67] and atrial fibrillation [68]. New biomarkers can be developed by utilizing such methodologies and provide stepping stones for new therapy options. DNA microarray experiments involving animal models have been performed to understand the Brown Norway to Lewis heterotopic heart transplant model [69]. A recent study has documented a correlation between endomyocardial biopsy material and whole blood by using microarray analysis. A new biomarker panel from whole blood samples is proposed for detection of rejection, which was claimed to have as efficient results as biopsy microarray [70].

Despite the major improvements that have been achieved in the prevention and treatment of acute transplant rejection, long-term survival following HT has not improved. Cardiac allograft vasculopathy (CAV) is the major long-term limitation of HT survivors [71, 72]. Despite a 2–4 % recent decrease in the cumulative incidence of CAV, the prevalence of CAV still remains high. CAV is detectable by angiography in 8 % of the patients within the first year, 20 % at 3 years, 32 % at 5 years, and 45 % at 8 years following HT [21]. Briefly, CAV is the leading cause of late morbidity and mortality in HT patients, accounting for 30 % mortality in the first 5 years [21]. The pathogenesis of CAV is not fully elucidated. Indeed, CAV is a complex, multifactorial phenomenon. Immunologic (indirect allorecognition, upregulation of cytokines and cytokine-related adhesion molecules, focal inflammation, and vasculitis) and nonimmunologic factors (repetitive vascular injury) may play critical roles in the process [32, 73–75].

EMB and coronary angiography provide effective but invasive monitoring methods for CAV in cardiac transplantation recipients. Histologically CAV is characterized by a concentric and diffuse intimal thickening composed of T cells, macrophages, and modified smooth muscle cells beneath an activated endothelium in epicardial and intramural coronary arteries along their entire lengths [76–83]. The disease begins as concentric fibrous intimal thickening caused by myofibroblast proliferation and fibrosis mainly in the proximal region of epicardial arteries [80]. Following the first year of HT, intermediate lesions with intimal lipid-filled cell accumulation can be detected. In long-term survivors, fibrous and fibrofatty intimal lesions often diffusely involve large and small epicardial and intramural arteries [80–83]. Late increase in TNF-α and IFN-γ in endomyocardial biopsies precedes the ­development of CAV [84]. van Loosdregt and colleagues demonstrated abundant CD4+ T cells that express HLA DR in transplanted hearts with CAV [85]. IL-17 has been shown to be critical in TGFbeta-driven allograft fibrosis [86]. Recently, elevated levels of circulating smooth muscle progenitor cells (SPCs), but not EPCs, and elevated plasma CXCL12 concentrations were found in CAV patients in contrast to patients without CAV [87]. Furthermore, the presence of coronary inflammatory plaque as assessed by virtual histology intravascular ultrasound (VH-IVUS) was associated with early recurrent rejection and with subsequent progression of CAV [88]. Metabolic syndrome is associated with endothelial dysfunction and increases the risk of CAV [89]. New-generation immunosuppressive drugs and immunomodulatory medication may have beneficial effects on coronary microvasculature following heart transplantation [90].