Based on the concepts of systems biology, this study was designed to identify new prognostic biomarkers after acute MI. First, using a test cohort of 32 patients, we observed that LV dysfunction after MI (4-months EF < 40%) had a biosignature in blood cells in the acute phase. Among the genes associated with LV dysfunction, 28 were linked to angiogenesis, which is known to play a key role in cardiac repair. Then, using clustering analysis of a network of protein-protein interactions built with these 28 angiogenic genes, we isolated a group of 3 genes -TGFBR1, PTK2, ITK- which predicted LV function with and AUC of 0.89. In an independent validation cohort of 115 MI patients, TGFBR1 was found to have a prognostic value which may become clinically useful. Finally, we showed that the TGFB1-TGFBR1 is activated after MI in rats and correlates with the extent of remodeling.
Left ventricular remodeling is a highly complex phenomenon involving diverse biological processes, such as inflammation, regulation of extracellular matrix turnover, fibrosis, cell death, and angiogenesis. Such complex diseases can be approached with systems-based concepts. We chose angiogenesis to filter the relatively high number of genes (525) found to be differentially expressed between patients with or without LV dysfunction. It is clear that other family of genes involved in remodeling may also be worth studying. It is expected that genes involved in multiple biological pathways or sharing many interactions with other genes may be more susceptible to play significant roles in remodeling and have a prognostic value. Accordingly, TGFBR1 was a node in the network of 16 angiogenic proteins shown in Figure 2B and was connected to 6 other proteins, suggesting a central role in angiogenesis. A more global approach, which could be used for future studies, would be to widen the search for biomarkers to all pathways known to play a role in LV remodeling, and not to restrain to a specific pathway such as angiogenesis.
Transcriptional profiling has emerged as an interesting tool to study cardiovascular diseases and ultimately to personalize therapeutic strategies . A key feature of our study is the use of readily available blood cells, rather than cardiac biopsies. While it may be argued that transcriptomic analysis of cardiac tissues would more accurately reflect the myocardial response to MI, it is accepted that several cardiovascular conditions including coronary artery disease  and chronic HF  are characterized by specific transcriptomic biosignatures in blood cells. Our study shows for the first time that TGFBR1 expression level in blood cells obtained at presentation in acute MI patients has prognostic value for long-term LV remodeling.
In addition, we have observed an association between TGFB1, which has prognostic values for remodeling and hypertrophy in patients with hypertension and aortic stenosis [18, 19], and LV function. This association was not as robust as TGFBR1, and the prognostic value of TGFBR1 was not found to be improved by TGFB1 determination. Interestingly, TGFBR1 added some value to the prediction of TnT, a marker of infarct size known to affect LV remodeling. The reclassification of 8.2% of patients misclassified by TnT is clinically relevant. Similarly, the reclassification of 17.7% of patients misclassified by a standard risk factor model based on age, gender and time to reperfusion, is pertinent.
The integration of multiple biomarkers is thought to improve the estimation of the course of cardiovascular diseases , although this assumption has been questioned . Heidecker and coworkers reported that a group of 45 genes identified from endomyocardial biopsies predicted 5-year outcome of new onset HF patients with a sensitivity of 74% and a specificity of 90% . More recently, a panel of 5 plasma proteins involved in extracellular matrix turnover was demonstrated to be a more accurate predictor of LV hypertrophy than any single biomarker . Alternatively, combining biomarkers representing diverse disease mechanisms has also been shown to add incremental risk stratification value in patients with non ST-elevation acute coronary syndrome . Our data showing that TGFBR1 has an additive value to traditional markers are consistent with the concept that multiplication of biomarkers may be clinically useful.
More than 80% of the patients enrolled in this study were males. The low number of females enrolled (6 in the test cohort and 17 in the validation cohort) prevented us from investigating whether gender contributes to the prognostic value of TGFBR1. However, TGFBR1 expression was not different between women and men (not shown), suggesting that gender may not influence the prognostic value of TGFBR1.
Activation of the TGFB1-TGFBR1 pathway is mainly associated with stimulation of fibrosis, although it also affects LV hypertrophy, matrix metabolism, inflammation, and angiogenesis [25–27]. After MI, this pathway triggers the switch from inflammation to fibrosis [28–30]. TGFBR1 expression has been reported to be higher in the infarcted region of pig hearts after permanent coronary ligation compared to the remote region . Our data in rats confirm and extend these observations. It is believed that biomarkers should reflect the pathophysiology, and our experiments in rats provide valuable proof for this. Inhibition of TGFBR1 activity by orally active specific inhibitors [28, 32] or competitive inhibition of TGFB1 by a soluble form of TGFBR2  dampens cardiac remodeling after MI. Other strategies to block TGFB, which have been recently reviewed , may also be tested to limit cardiac remodeling
Endothelial progenitor cells participate in cardiac revascularization and healing after acute MI. However, considering their extremely low frequency in the circulation, these cells are certainly not the main source of TGBFR1 signal measured by microarrays. Circulating monocytes-macrophages may most probably account for the majority of TGFBR1 expression. Interestingly, these cells secrete many cytokines, growth factors and fibrotic factors which regulate LV remodeling and cardiac healing. The higher expression of TGFBR1 in patients with low EF is consistent with the more robust activation of inflammation observed in these patients, as attested by higher white blood cells counts (not shown). Whole blood cells profiling can be affected by shifts in leukocyte populations. However, the proportions of circulating monocytes were comparable between patients with low EF and patients with high EF, suggesting that this confounding factor did not importantly affect our results. It would be interesting to accurately determine which cell type(s) express TGFBR1 in the heart, and whether TGFBR1 expression is regulated in cells involved in cardiac healing. The animal model of coronary artery ligation used in the animal study is a valuable tool to answer these questions.
The main limitation of the present study relies in the small number of patients enrolled. This is particularly relevant for the test cohort used in microarray experiments. Nevertheless, using a different and quantitative technique, we were able to validate our findings from this small cohort in an independent cohort of more than 100 patients. The relevance of our findings depends on the confirmation of the prognostic value of TGFBR1 in larger patient populations. In addition, all human blood samples were collected at presentation and measurement of TGFBR1 expression at different time-points after acute MI could provide valuable informations. Finally, mutations in TGFBR1 gene, known to affect vascular integrity in Marfan and Loeys-Dietz syndromes [35, 36], have to be taken into consideration for the design of probes to measure TGFBR1 expression and for the design of specific therapeutic inhibitors.