P2Y₁₂-dependent activation of hematopoietic stem and progenitor cells promotes emergency hematopoiesis after myocardial infarction

Inflammation is an essential prerequisite for wound healing and cardiac remodeling after myocardial infarction (MI) [43]. Acute ischemic myocardial injury triggers a systemic inflammatory response including innate immune cell production and release, local recruitment, maturation and apoptosis [19, 36, 50]. The infiltrating immune cells orchestrate breakdown and removal of necrotic debris as well as lesion repair with collagen deposition and neovascularization [44, 78]. These changes arise in the necrotic area but also stretch to the borders of the infarcted myocardium and even remote zones [34]. However, excessive inflammation or inadequate resolution of the inflammatory response after MI may advocate adverse cardiac remodeling and accelerate heart failure [47, 58].

Injured cardiac cells secrete various chemoattractants that have been appreciated to locally regulate post-MI inflammation [5, 15]. In addition, activated platelets release a plethora of prothrombotic factors and immunoregulatory cytokines to promote endothelial activation and facilitate platelet-leukocyte-complex formation [9, 22, 70]. Following the resulting cytokine gradients, immune cells are recruited to the site of injury [5]. As a consequence of the high leukocyte turnover after MI, reservoirs of preformed innate immune cells in the bone marrow (BM), spleen and blood are rapidly depleted and rely on immediate resupply by emergency hematopoiesis to compensate for the excessive demand [36, 49].

Upstream in the hematopoietic hierarchy, hematopoietic stem and progenitor cells, defined as LSK cells based on their characteristic surface expression pattern (Lineage ^neg, Sca-1⁺, c-Kit⁺) [42], are activated and enter the cell cycle to increase the production of myeloid lineage progeny, predominately [11]. Multiple cascades have previously been described that transport information from the site of injury to the BM [65], involving blood borne factors, e.g. interleukin-1β (IL-1β) and interferons [3, 12, 57] or extravascular sympathetic nervous signaling [6, 10, 25]. IL-1β was shown to activate emergency hematopoiesis both directly by acting on LSK and indirectly by downregulating retention factors in the hematopoietic BM niche that are necessary for HSC homeostasis [57]. Likewise, noradrenaline released by sympathetic nerve fibers decreases the retention factor CXCL12 in the BM niche, subsequently elevating LSK proliferation [25].

Importantly, extracellular nucleotides, e.g. adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP) and uridine-5′-diphosphate (UDP), represent another subset of soluble danger signals after myocardial injury that activate purinergic receptors [24, 29, 46]. In cardiovascular disease, ADP is a key regulator of platelet activity and inhibition of the purinergic receptor P2Y₁₂ has thus emerged as an important therapeutic strategy to reduce recurrent cardiovascular events [72, 75].

Intriguingly, LSK cells have been reported to also express purinergic receptors [13, 56]. While purinergic P2X receptors on hematopoietic progenitors have recently been described to impact cell trafficking, the role of ADP-sensitive P2Y receptors has not yet been characterized [1].

In this study, we show that ADP acts as danger signal for the hematopoietic BM after MI and fosters emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression in LSK via P2Y₁₂-dependent signaling. Using platelet-specific as well as global P2Y₁₂-deficiency models and treatment with the potent P2Y₁₂ inhibitor prasugrel, we demonstrate that P2Y₁₂ inhibition reduces emergency hematopoiesis and the excessive inflammatory response to MI, subsequently preserving cardiac function and preventing adverse cardiac remodeling after MI. Targeting the ADP-dependent, P2Y₁₂ receptor-mediated signaling pathway after MI may thus exert beneficial, non-canonical effects beyond inhibition of platelet activation.

After MI, blood leukocyte counts correlate with in-hospital mortality and recurrent adverse cardiovascular events [21, 23]. Modulating the inflammatory response to ischemic myocardial injury has therefore been a promising approach to improve patients’ outcome after MI and has recently been in focus of several clinical trials on secondary cardiovascular prevention [45, 54, 55, 67]. In this study, we describe that the ADP-dependent, P2Y₁₂ receptor-mediated signaling pathway is a key driving factor for emergency hematopoiesis after MI. We identified ADP as a danger signal for the hematopoietic BM since ADP levels increased after MI and promoted phosphorylation of Akt and cell cycle progression of hematopoietic stem and progenitor cells (LSK) in vitro. We detected P2Y₁₂ receptor expression on LSK cells which implicates that ADP acts directly on LSK cells via P2Y₁₂ signaling, not mediated by P2Y₁₂ on platelets, which was confirmed in mice with platelet-specific P2Y₁₂-deficiency. Ubiquitous P2Y₁₂ knockout or treatment with the P2Y₁₂ receptor antagonist prasugrel modulated emergency hematopoiesis, subsequently reducing the excessive inflammatory response to MI, which translated into reduced expansion of downstream lineages and limited the numbers of leukocytes in circulation and in the infarct. Ultimately, this preserved cardiac function and prevented adverse cardiac remodeling after MI (Fig. 7).

Acute ischemic myocardial injury triggers a profound release of danger-associated molecular patterns (DAMPs) and cytokines from activated platelets, injured cardiomyocytes and endothelial cells [51]. These danger signals then initiate a pro-inflammatory cascade that mediates local inflammation, steers leukocyte recruitment to the infarct [15, 20] and fosters emergency hematopoiesis in the bone marrow to meet the excessive demand for leukocytes [3, 6, 10, 12, 25, 65]. Extracellular nucleotides such as ATP, ADP and UDP are among the prominent danger signals released upon vascular injury [7]. Whereas ATP is most abundant upon hypoxia or tissue acidosis and can activate several P2 receptors [51, 74], ADP is primarily known to be secreted from platelets’ dense granula upon activation and to act as a ligand to the purinergic P2Y₁₂ receptor. Purinergic P2 receptors are closely linked to the inflammatory cascade [24, 61]. Intriguingly, hematopoietic stem and progenitor cells have been reported to express P2X and P2Y receptors in mice and humans [13, 17, 35, 56, 73]. While P2X₇ has previously been linked to mobilization and homing of hematopoietic stem cells [1], the role of the P2Y receptors on hematopoietic progenitors, especially the P2Y₁₂ receptor, has not yet been characterized.

Most DAMPs are released instantly in the course of injury. Yet, we found ADP levels in the BM to peak specifically on day 2 after MI. Interestingly, this increase in ADP concentrations coincided with the onset of hematopoietic stem cell activation following ischemic myocardial injury as previously reported [10, 11, 57], suggesting that ADP possibly serves as a messenger to the BM in favor of emergency hematopoiesis. Indeed, stimulating hematopoietic BM cells with ADP in cell culture increased BM cell proliferation. Importantly, platelets were excluded during the cell selection process for this experiment as ADP-dependent platelet activation in culture could have confounded data via cytokine release.

Looking for possible sources of ADP in the BM, we primarily considered activated platelets as well-acknowledged origin of extracellular ADP in hemostasis. Platelet inhibition, however, did not significantly decrease ADP concentrations in the BM or blood. As an alternative source, conversion of ATP to ADP and AMP, which occurs within hours after release, could be discussed as a delayed source of ADP as a secondary messenger [64]. However, detecting ATP conversion to ADP after ischemia has been impeded due to the short half-life of ADP and ample presence of ectonucleotidases in vivo [53, 77], leaving the designated origin of ADP in the hematopoietic BM after MI still to be elucidated.

To identify possible recipients to the elevated ADP levels, we screened various hematopoietic cell populations for the expression of the most commonly known ADP receptor P2Y₁₂. Beyond the well appreciated P2Y₁₂ expression on platelets [28], several other cell types including microglia in the brain, osteoclasts, vascular smooth muscle cells, leukocytes and hematopoietic progenitor cells have been reported to express P2Y₁₂ [8, 18, 63, 73]. However, the functional relevance of P2Y₁₂ expression beyond platelet aggregation is largely unknown [14]. Remarkably, after careful exclusion of platelet-complexes in the cell isolation process, we detected P2Y₁₂ receptor expression, specifically on hematopoietic stem and progenitor cells (LSK) in the murine BM. This finding could be validated interrogating an external RNA sequencing dataset of isolated LSK cells [33]. In line with previous studies, we were also able to detect P2Y₁₂ expression on differentiated leukocyte subsets at lower expression levels as compared to LSK cells. Therefore, direct P2Y₁₂ dependent effects on leukocyte subsets have to be acknowledged and remain to be investigated.

The phosphoinositide 3-kinase (PI3K)/Akt intracellular signaling pathway promotes cell survival, proliferation and growth by phosphorylation and inhibition of key transcription factors and has been reported to forward Akt phosphorylation downstream of the P2Y₁₂ receptor in activated platelets and vascular smooth muscle cells [32, 48, 59]. Stimulating LSK cells with ADP, we detected increased Akt phosphorylation, which was absent in P2Y₁₂-deficient LSK cells. Furthermore, performing LSK-specific CFU assays confirmed LSK as a protagonist cell population. The P2Y₁₂ receptor was validated to primarily moderate the expansion of hematopoietic BM cells in response to ADP, as P2Y₁₂-deficiency depleted the pro-proliferative effect of ADP. This minimized possible pleiotropic effects due to dephosphorylation of ADP to AMP and adenosine to be responsible for the effect on hematopoietic BM proliferation. Taken together, our experiments implicate a close relation of ADP-dependent P2Y₁₂ signaling with Akt phosphorylation and cell cycle progression in LSK cells and appeared to be well in line with previous reports on Akt-dependent proliferation of LSK cells with primarily myeloid lineage differentiation [31].

We performed permanent coronary ligation to induce acute ischemic myocardial injury in a murine model to analyze the inflammatory response to MI in the setting of selective P2Y₁₂ targeting, achieved by P2Y₁₂-deficiency or pharmacological P2Y₁₂ receptor inhibition. Permanent coronary ligation was preferred over an ischemia / reperfusion model to reduce the effect of local, platelet-mediated reperfusion injury after MI. For P2Y₁₂ antagonist treatment, we chose the second generation thienopyridine prasugrel, which was reported to be more potent than clopidogrel in terms of more rapid and consistent P2Y₁₂ inhibition [75]. Despite its promising favorable effects on infarct size, cytokine release and cardiac remodeling [38, 69, 72] the competitive P2Y₁₂ inhibitor ticagrelor was not used due to known pleiotropic effects beyond P2Y₁₂ receptor inhibition, i.e. inhibition of toll like receptors-1/2 (TLR1/2), the protease activated receptor (PAR)-pathway [71] and the equilibrative nucleoside transporter 1 (ENT1), which increases extracellular concentrations of adenosine [2]. Ubiquitous P2Y₁₂ knockout or prasugrel treatment both attenuated hematopoietic BM activation after MI, reflected in reduced cell cycle activity of LSK cells, which transferred to lower numbers of downstream progenitors and leukocytes of myeloid and lymphocyte origin in the blood. Intriguingly, neutropenia has been described as an adverse drug reaction in some patients treated with P2Y₁₂ inhibitors and, vice versa, a small but significant rise in blood neutrophil counts after terminating P2Y₁₂ antagonist therapy has been recorded [62, 75]. Next to myeloid cells, lymphocytes are further key players in the remodeling of cardiac lesion [26, 27]. Hence, P2Y₁₂ inhibition seems to not only modulate innate but also adaptive immunity after MI.

Scaled down emergency hematopoiesis and reduced leukocytosis after MI by P2Y₁₂ inhibition with prasugrel translated to limited leukocyte infiltration of primarily myeloid origin to the infarct, ameliorated adverse cardiac remodeling and preserved cardiac function after MI. Notably, P2Y₁₂ receptor inhibition resulted in higher collagen content in the infarct border zone. While exaggerated post-MI cardiac fibrosis especially outside the infarct zone has been associated with reduced cardiac compliance [66], locally restricted increase of the collagen portion in the border zone may also indicate a well-balanced wound healing with increased tissue stability. Supported by signs of neovascularization, P2Y₁₂ inhibition post-MI preserved cardiac function and reduced adverse cardiac remodeling.

Crosstalk between the injured myocardium and hematopoietic BM uses multiple channels, moderated by soluble danger signals such as pro-inflammatory interleukin-1β and the sympathetic nerve system (SNS). [6, 25, 57] BM retention factors, namely CXCL12, VCAM1, SCF and angiopoietin, are secreted by regulatory BM niche cells and modulate LSK homeostasis [42]. Both IL-1β and SNS induce downregulation of BM retention factors to activate LSK cells [10, 57]. Putting our findings in the context of the appreciated signaling pathways that promote emergency hematopoiesis after MI, we evaluated cornerstones of IL-1β-mediated and sympathetic nervous signaling under the treatment with P2Y₁₂ antagonist prasugrel. Plasma levels of IL-1β after MI as well as expression of BM niche factors were not affected by P2Y₁₂ inhibition with prasugrel. Also, the release of LSK and progenitor cells from the BM into circulation, previously described to play an essential role for IL-1β-dependent extramedullary hematopoiesis after MI [57], remained unchanged by prasugrel treatment and supports the hypothesis of a direct ADP-dependent and P2Y₁₂ receptor-mediated effect on LSK cells.

In the setting of ischemic myocardial injury and pressure overload [39, 76], P2Y₁₂ receptor inhibition alleviates adverse cardiac remodeling and preserves cardiac function via platelets’ immunoregulation, closely linked to leukocyte recruitment with platelet-leukocyte, platelet-endothelial and enhanced leukocyte-endothelial interactions [16, 39]. In this context, P2Y₁₂ inhibition was shown to limit platelet-leukocyte-conjugation by reducing platelet p-selectin expression [39], preventing leukocyte rolling for transendothelial migration [70]. To distinguish our findings from platelet-P2Y₁₂, we analyzed emergency hematopoiesis in the acute phase after MI in platelet-specific P2Y₁₂-deficient GFP chimera. The UBC-GFP reporter allowed us to rule out residual presence of P2Y₁₂-competent platelets in recipient mice. As before, prasugrel treatment still reduced LSK cycling in the bone marrow and leukocyte numbers in circulation and the infarcted myocardium in the acute phase after MI, indicating a significant role of P2Y₁₂ signaling in inflammation beyond platelet-induced immunoregulation. Furthermore, acetylsalicylic acid (ASA) which inhibits p-selectin expression on platelets, exerted no additional effect on emergency hematopoiesis [30, 41].

Translational investigation of P2Y₁₂ signaling in emergency hematopoiesis after MI is limited by the otherwise widely appreciated benefits of P2Y₁₂ inhibitors in cardiovascular disease [68]. Yet, there are intriguing observations in support of enhanced anti-inflammatory properties of P2Y₁₂ inhibitors. Adding P2Y₁₂ antagonists to ASA, considered as dual antiplatelet therapy (DAPT), proved to be superior to any other combination in stable and unstable coronary disease [37, 68]. This benefit was not limited to the expected prevention of target vessel failure but also reduced reoccurring adverse cardiac events [4].

In our study, we describe a novel pathway in the crosstalk of injured myocardium and the hematopoietic BM after MI, fostering emergency hematopoiesis via ADP-dependent P2Y₁₂ receptor-mediated stimulation of upstream hematopoietic stem and progenitor (LSK) cells. Inhibition of the P2Y₁₂ receptor modulated the inflammatory response to injury, preserved cardiac function and prevented adverse cardiac remodeling after MI. Given the high demand and turnover of leukocytes following MI [36], the modulation of emergency hematopoiesis may thus be an intriguing approach to target inflammation at its root, prevent excessive secondary myocardial damage and sustain cardiac function.