Increased vascularisation supports heart recovery after ischemia. The formation of new vessels in the hypoxic area restores blood flow, provides oxygen and nutriments to the surviving cells, and promotes the migration and engraftment of new cells. While angiogenic inhibition contributes to the development of heart failure in cardiac injury animal models, early heart reperfusion or increased angiogenesis improves cardiac function and delays the onset of heart failure in patients suffering from cardiac ischemia (Shiojima et al., 2005; Friehs et al., 2006; Tirziu et al., 2007).

Angiogenesis is the main mechanism of neovascularisation in adult infarcted hearts (Manavski et al., 2018; Li et al., 2019; Ray et al., 2010). The origin of new endothelial cells (i.e. resident or infiltrating) as well as the underlying mechanism leading to their proliferation (partial endothelial-to-mesenchymal transition [EndMT] or not) have long been debated. The current consensus is that after myocardial infarction (MI), angiogenesis in the infarct border zone of the heart occurs by clonal expansion of pre-existing resident endothelial cells with no EndMT mechanism (Manavski et al., 2018; Li et al., 2019; He et al., 2017). However, the molecular pathway involved in myocardial neovascularisation after ischemia remains unknown. Indeed, Payne et al., 2019 recently demonstrated that the developmental VEGFA-MEF2 pathway, which was thought to be involved, is in fact impaired in adult ischaemic hearts.

Stimulating angiogenesis after MI can improve heart recovery. One complementary therapy could be ‘re-activating’ vasculogenesis (i.e. the differentiation of precursor cells into mature endothelial cells), a mechanism that occurs in the heart during development but is quiescent in adult hearts. Epicardial cells, and more precisely, cells expressing the Wilms’ tumour 1 transcription factor (WT1) migrate from the epicardium to the myocardium during heart development and then differentiate into coronary endothelial cells, pericytes, smooth muscle cells, and even cardiomyocytes after epithelial-to-mesenchymal transition (Zhou et al., 2008; Cano et al., 2016; Smits et al., 2018). Consequently, numerous WT1⁺ cells are found in foetal and neonatal hearts, whereas only a few cells express WT1 in adult hearts (Duim et al., 2016; Duim et al., 2015).

In hypoxic adult hearts, numerous proliferating WT1⁺ cells can be localised in the epicardium near the infarct and border zone, suggesting that hypoxia stimulates either the proliferation of the remaining WT1⁺ cells or the re-expression of WT1 in cardiac cells (Duim et al., 2015; Balbi et al., 2019; Zhou et al., 2012). WT1⁺ epicardium-derived cells (EPDCs) remain in a thickened layer on the heart surface without migrating into the myocardium or differentiating into other cell types such as cardiomyocytes or endothelial cells (Zhou et al., 2011). Different treatments (e.g. injections of thymosin beta four or human amniotic fluid stem cell secretome) fail to induce WT1⁺ cell differentiation into endothelial cells in adult hearts after MI (Balbi et al., 2019; Zhou et al., 2012). Despite enhanced WT1⁺ cell proliferation and higher vessel density in these treated infarcted hearts, no WT1⁺ cell differentiation into endothelial cells has been detected, implying that WT1⁺ EPDCs improve neovascularisation in infarcted hearts via paracrine stimulation.

It is therefore important to identify soluble factors to increase neovascularisation in the heart after MI. For several years, we have studied the role of brain natriuretic peptide (BNP) in the heart during ageing and after ischaemic damage (Bielmann et al., 2015). BNP is a cardiac hormone belonging to the natriuretic peptide family along with atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP). Although low levels of BNP are co-stored with ANP in atria, high levels of BNP are detected in the ventricle (Potter et al., 2009). BNP is mainly secreted in the ventricles by cardiomyocytes, fibroblasts, and endothelial and precursor cells (Bielmann et al., 2015; Potter et al., 2009; Rosenblatt-Velin et al., 2016). BNP binds to guanylyl cyclase receptors, NPR-A and NPR-B, thus increasing the intracellular cGMP level (Potter et al., 2009).

BNP is synthesised in the cell cytoplasm as pre-proBNP precursor, cleaved by furin and corin into proBNP peptide, and then into the biologically active carboxy-terminal BNP peptide (active BNP) and the inactive N-terminal fragment (NT-proBNP) (Yandle and Richards, 2015; Clerico et al., 2012). ProBNP, active BNP, and NT-proBNP peptides are continuously secreted by cardiac cells and ProBNP peptide is the major circulating form of BNP in the plasma of healthy individuals (Shimizu et al., 2002). O-glycosylated proBNP was also detected in the plasma of patients suffering from heart failure (Volpe et al., 2016), and O-glycosylation of proBNP prevents cleavage of this peptide (Volpe et al., 2016). Thus, the balance between proBNP and active BNP is impaired in patients with heart diseases, as they have higher levels of inactive proBNP and reduced levels of active BNP. Patients with heart disease therefore have a deficit in functional active BNP (Chen, 2007).

BNP secreted by cardiac cells acts on different organs such as the kidneys (modulating sodium and water excretion), vessels (increasing dilation), fat (increasing lipolysis), and pancreas (modulating insulin secretion) (Rosenblatt-Velin et al., 2016; Volpe et al., 2016). In the heart, a majority of cardiac cells (cardiomyocytes, fibroblasts, endothelial cells) express BNP receptors in physiological and pathological states (Bielmann et al., 2015). BNP treatment after injury protects the heart by reducing fibrosis, cardiomyocyte death, and hypertrophy (D'Souza and Baxter, 2003; Moilanen et al., 2011; Gorbe et al., 2010; Scott et al., 2009; Sun et al., 2010; Wu et al., 2009).

In the last few years, we and others reported that BNP supplementation after ischaemic damage promotes the recovery of cardiac function and prevents cardiac remodelling in adult rodent ischaemic hearts (Bielmann et al., 2015; Moilanen et al., 2011). Furthermore, in clinic, a treatment (LCZ696 or Entresto, Novartis) based on inhibition of neprilysin, an enzyme involved in the degradation of the natriuretic peptides, leads to reduced rate of mortality in patients suffering from heart failure with reduced and preserved cardiac contractility or ejection fraction (McMurray et al., 2014). The cellular mechanisms by which BNP exerts its cardioprotective effect are not fully elucidated (Bielmann et al., 2015; Rosenblatt-Velin et al., 2016; Moilanen et al., 2011). Curiously, we observed that BNP stimulates in vitro and in vivo (i.e. in adult infarcted hearts) the proliferation of cardiac non-myocyte cells (NMCs) expressing the stem cell antigen-1 (Sca-1) (Rignault-Clerc et al., 2017). As Sca-1⁺ cells in adult hearts were reported to be pure endothelial cells (Zhang et al., 2018), we questioned whether BNP modulates endothelial cell fate.

Thus, since high BNP levels in plasma are associated with increased collateral development in patients with coronary artery disease (Xi et al., 2011), this work aimed to determine whether part of the cardioprotective effect of BNP treatment after experimental MI involves increased neovascularisation.

The work presented here continues our previous research aimed at determining the role of BNP in the heart. We already demonstrated that BNP injections in mice after MI decreased heart remodelling and increased heart function (Bielmann et al., 2015). We thus questioned whether increased neovascularisation in infarcted hearts is part of the cardioprotective effect of BNP.

In this study, we first showed that BNP treatment increases myocardial vascularisation and the number of endothelial cells in the infarct zone and border zone (ZI+BZ) as a well as in the remote zone (RZ) of infarcted hearts. Second, BNP stimulates the proliferation of endogenous pre-existing endothelial cells, likely via NPR-A binding and p38 MAP kinase activation. Third, BNP stimulates and/or accelerates the re-expression of the WT1 transcription factor in cardiac cells after MI. Fourth, in the infarcted area of untreated injured hearts, WT1⁺ EPDCs proliferate and modestly contribute to heart neovascularisation by differentiating into endothelial cells. Lastly, BNP stimulates WT1⁺ EPDC proliferation, with more endothelial cells originating from WT1⁺ EPDCs in BNP-treated infarcted hearts.

This is the first work to demonstrate that intraperitoneal injections of BNP increase neovascularisation of the heart after MI. Thus, part of the cardioprotective effect of BNP in infarcted hearts is probably due to accelerated and/or increased neovascularisation in both the ZI+BZ and RZ. This result corroborates works reporting that natriuretic peptides stimulate angiogenesis and vasculogenesis during development and in adult ischaemic organs (Moyes and Hobbs, 2019; Kuhn, 2012). ANP via NPR-A stimulates endothelial precursor cell proliferation, migration, and differentiation in the skin during cutaneous wound healing (Lee et al., 2018). The restoration of blood flow is impaired after hindlimb ischemia in NPR-A KO mice (Kuhn et al., 2009), while the injection of a high concentration of CNP increases vascular density in infarcted swine hearts (Del Ry et al., 2013). Recently, CNP was identified as a key regulator in angiogenesis and vascular remodelling after ischemia in patients suffering from peripheral artery disease (Bubb et al., 2019). Other findings reported that BNP injections in mice lead to increased vascular regeneration in ischaemic limbs (Shmilovich et al., 2009) and that intramyocardial BNP gene delivery via adenovirus increased capillary density in normal rat hearts but not in infarcted hearts (Moilanen et al., 2011). Our article makes a novel contribution by showing the ‘time- and area-dependent’ effect of BNP and the identification of both different mechanisms by which BNP increases the number of endothelial cells in infarcted hearts.

In our mouse model, the timing and action mechanisms of BNP stimulation differed in the myocardium in the ZI+BZ and RZ of infarcted hearts. In the RZ of infarcted hearts, the number of endothelial cells and vascularisation increased 3 days after MI. In the ZI+BZ, we detected more endothelial cells 3 days after MI but vascularisation increased only 10 days after MI. BNP stimulates endothelial cell proliferation first in the RZ (1–3 days after MI induction) and then in the ZI+BZ (10 days after MI). Although BNP seems to induce the re-expression of the WT1 transcription factor in cardiac cells in both areas of infarcted hearts (3 to 10 days after MI), its effect on WT1⁺ EPDCs is limited to ZI+BZ of infarcted hearts 10 days after MI.

The delay in stimulating endothelial cell proliferation in the different areas of infarcted hearts can be explained by the bioavailability of BNP (less cell deaths, more vessels and capillaries in the RZ), but its effect on WT1⁺ EPDCs must be favoured by the microenvironment and probably hypoxia. Indeed, in the ZI+BZ, cells undergo a synergistic effect of hypoxia and BNP.

Concerning the mechanisms, we showed that BNP acts directly on mature endothelial cells by stimulating their proliferation. The fact that BNP treatment did not increase the number of endothelial cells in neonatal NMCs isolated from neonatal NPR-A deficient hearts in our study demonstrates that BNP acts via NPR-A on neonatal cardiac cells. In the mouse model of hindlimb ischaemia, proliferating satellite cells secreted BNP, which stimulates angiogenesis in the neighbouring endothelium cells (Kuhn et al., 2009). This mechanism is also impaired in NPR-A KO mice, suggesting that regardless of the organ, BNP stimulates mature endothelial cell proliferation via the NPR-A receptor.

In our work, BNP treatment modulated also the fate of immature cells such as WT1⁺ EPDCs. This is not the first work to report the effect of BNP on immature or precursor cells. BNP level is highly correlated with the number of circulating endothelial precursor cells in patients suffering from heart failure (Shmilovich et al., 2009). In vitro, the proliferation, adhesion, and migration capacities of endothelial precursor cells increased in a dose-dependent manner after BNP treatment (Shmilovich et al., 2009). Furthermore, BNP stimulated the proliferation of satellite cells in a hindlimb ischemia model, which produced endogenous BNP stimulating the regeneration of endothelial cells (Kuhn et al., 2009).

Among cells expressing the WT1 transcription factor, it is important to discriminate between true WT1⁺ EPDCs and cardiac cells re-expressing the WT1 transcription factor in ischaemic hearts (Duim et al., 2015). While the function of WT1 re-expression in infarcted hearts is poorly understood, it seems necessary for endothelial cell proliferation (Duim et al., 2015), with our results showing that BNP treatment increases and/or accelerates this process. However, in Cdh5:ROSA infarcted hearts, we detected GFP⁺ WT1⁺ BrdU⁺ cells (i.e. endothelial proliferating cells expressing WT1) as well as GFP⁺ WT1^- BrdU⁺ cells (i.e. endothelial proliferating cells without WT1 expression), which shows that WT1 expression is transient or unnecessary for endothelial cell proliferation.

Regarding WT1⁺ precursor cells, we induced MI in WT1:ROSA hearts and analysed the fate of GFP⁺ cells. We observed the proliferation of WT1⁺ EPDCs, mainly localised in the subepicardial layer after MI as previously reported (Duim et al., 2015; Balbi et al., 2019; Zhou et al., 2012). Although the differentiation of WT1⁺ cells into endothelial cells in infarcted hearts is still debated (Zhou et al., 2008; Zhou et al., 2012; Zhou et al., 2011), we found that 5% of endothelial cells in the ZI+BZ originated from WT1⁺ EPDCs in saline-treated infarcted hearts. Interestingly, 50% of WT1⁺ cells differentiated into endothelial cells in these infarcted hearts. This shows that WT1⁺ EPDCs have the natural capacity to differentiate into endothelial cells in adult hypoxic hearts, as is the case during embryogenesis (Zhou et al., 2008). Due their low number, it was nevertheless difficult to highlight this process.

BNP increased the number of WT1⁺ EPDCs in the subepicardial layer by stimulating their proliferation. Consequently, more cells migrated into the myocardium and differentiated into endothelial cells. We cannot exclude that BNP could also stimulate the migration of EPDCs into the myocardium, where the conditions could be adequate to differentiate into endothelial cells. Regardless of the mechanism, 10% of endothelial cells in the ZI+BZ originate from WT1⁺ EPDCs in BNP-treated hearts. However, the differentiation capacity of the WT1⁺ cells into endothelial cells was identical in saline- and BNP-treated hearts (about 50%). BNP therefore stimulates WT1⁺ EPDC proliferation but not their differentiation into endothelial cells.

Our study does not fully elucidate the signalling pathways by which BNP induces endothelial cell and WT1⁺ EPDC proliferation. Additional work is needed in this respect, especially regarding the link between BNP treatment and VEGF expression. Indeed, the reactivation of the VEGFA signalling pathway in adult infarcted hearts 10 days after surgery is likely the key of increased angiogenesis after BNP treatment. This pathway was shown to be inactive in adult ischaemic hearts, whereas it drives angiogenesis after ischemia in neonatal hearts (Payne et al., 2019). However, as some studies reported that natriuretic peptides repress VEGF synthesis (Pedram et al., 2001), it is unclear which mechanism(s) increased vegfa mRNA levels in both areas of the infarcted hearts 10 days after MI.

Interestingly, in our experiment, we did not detect increased vegfa mRNA levels in the RZ of infarcted hearts 1 or 3 days after MI, where the proliferation of endothelial cells increased. This suggests that, as shown by Kuhn et al., 2009 using a hindlimb ischemia model, BNP can also promote endothelial cell proliferation independently of VEGF secretion.

In the ZI+BZ, however, increased vegfa mRNA levels seem to induce the stimulation of WT1⁺ cell proliferation. Indeed, by stimulating WT1⁺ EPDCs with BNP, we obtained the same results as Zangi et al., who injected synthetic modified RNA encoding VEGF-A directly into the myocardium of infarcted mouse hearts, which stimulated WT1⁺ cell proliferation and shifted their differentiation into endothelial cells (i.e. 50% of WT1⁺ cells differentiated into endothelial cells) (Zangi et al., 2013). Thus, it seems that hypoxia and BNP increase VEGF, which stimulates WT1⁺ EPDC proliferation. The interactions between BNP and VEGF should therefore be studied in more detail to confirm whether BNP acts on endothelial cells via a VEGF-independent mechanism and on WT1⁺ EPDCs via a VEGF-dependent mechanism.

The findings presented in our study hold potential to offer new therapeutic strategies to improve the neovascularisation of hearts after MI or to aid neovascularisation in ischaemic hearts in patients with chronic coronary artery disease. Indeed, we identified BNP as a ‘new cardiac angiogenic’ factor, which stimulates the proliferation of both resident cardiac mature endothelial cells and WT1⁺ EPDCs. We have not yet identified the exact mechanisms by which BNP could act but re-activation of the VEGF-dependent pathway, active in infarcted neonatal hearts but inactive in adult infarcted hearts, may be possible (Payne et al., 2019). We also identified the proliferation and differentiation of WT1⁺ EPDCs as a mechanism, which can be targeted in infarcted adult hearts to increase heart revascularisation. To summarise, BNP binds to NPR-A to stimulate endothelial cell proliferation via p38 MAP kinase activation (Figure 10). BNP treatment also stimulates WT1⁺ EPDC proliferation. It remains to be determined whether BNP acts directly on these cells or via increased VEGF level (Figure 10). Interestingly, the benefit of LCZ696 treatment which reduces significantly the mortality of patients with chronic heart failure seems also to be associated with increased heart vascularisation, as a result of increased endothelial and WT1⁺ cell proliferation. However, whether this is associated with increased level of BNP remains to be demonstrated.

Figure 10

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All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Na Li

   Division of Angiology, Heart and Vessel Department, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland

   Contribution

   Software, Formal analysis, Project administration

   Competing interests

   No competing interests declared

2. Stephanie Rignault-Clerc

   Division of Angiology, Heart and Vessel Department, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland

   Contribution

   Software, Formal analysis, Project administration, perform animal experiments, flow cytometry analysis, molecular biology

   Competing interests

   No competing interests declared

3. Christelle Bielmann

   Division of Angiology, Heart and Vessel Department, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland

   Contribution

   Software, Formal analysis, Visualization, Methodology, perform immunohistology stainings, western blot analysis

   Competing interests

   No competing interests declared

4. Anne-Charlotte Bon-Mathier

   Division of Angiology, Heart and Vessel Department, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland

   Contribution

   Formal analysis, Methodology

   Competing interests

   No competing interests declared

5. Tamara Déglise

   Division of Angiology, Heart and Vessel Department, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland

   Contribution

   Software, Formal analysis, Methodology

   Competing interests

   No competing interests declared

6. Alexia Carboni

   Division of Angiology, Heart and Vessel Department, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland

   Present address

   Ecole Polytechnique fédéral de Lausanne, Laussane, Switzerland

   Contribution

   master student performing LCZ696 experiments

   Competing interests

   No competing interests declared

7. Mégane Ducrest

   Division of Angiology, Heart and Vessel Department, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland

   Contribution

   Formal analysis, medical student performing immunostainings on endothelial cells on LCZ696 treated mice

   Competing interests

   No competing interests declared

8. Nathalie Rosenblatt-Velin

   Division of Angiology, Heart and Vessel Department, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   nathalie.rosenblatt@chuv.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2011-2862

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