The homing of bone marrow stem cells is differentially activated in ischemic and valvular heart diseases and influenced by beta-blockers

Despite significant advances in therapeutic interventions, cardiovascular diseases (CVD) are still the leading cause of death worldwide. Among the CVD, ischemic heart disease (IHD) and non-ischemic valvular heart disease (VHD) are very frequent in older age. Despite significant improvement in the prognosis of these diseases, mortality rates remain high [1], mainly due to the limitation of conventional therapies to replace cardiomyocytes lost during cardiac ischemia [2].

Cellular cardiomyoplasty is an in situ implant of cells with the potential to induce cardiomyogenesis, angiogenesis and neovascularization, aiding the regeneration of damaged tissue [3]. In clinical practice, most studies have focused on the use of bone marrow-derived cells (BMC) in groups of patients with different heart diseases, describing a modest improvement in cardiac parameters, quality of life and patients’ symptoms [4]. Safety and effectiveness of therapy with BMC have also been demonstrated [5]. However, despite the well-designed, promising clinical studies, the mechanisms and molecules involved in cardiac repair and other issues that may optimize the beneficial effects of cardiomyoplasty remain to be clarified.

In cell therapy protocols, cell homing constitutes a critical step in the regeneration and repair process to promote cardioprotection and neoangiogenesis [6]. The level of expression of the chemokine SDF-1 and its receptor CXCR4 in stem cells influences the homing efficiency, so that the SDF-1/CXCR4 axis plays an essential role and offers potential advantages in cell therapy [7, 8]. Furthermore, CXCR7, another receptor with high affinity to SDF-1, is also involved in regenerating processes post-ischemic tissue [9].

Thus, considering the importance of cell homing to heart repair following injury, the present study aimed to assess the activation status of SDF-1/CXCR4 axes, in BM isolated from patients with IHD or VHD, target groups for stem cell therapy. We also analyzed the inflammatory profile and the influence of the use of some drugs, such as beta blockers, drugs widely used for these diseases, on the expression of the target molecules. Added to the existing knowledge about of stem cells homing, we can observe that the standard drug therapy used in cardiology, especially with beta-blockers, can interfere with the expression of molecules involved in this process, which should be considered in clinical trials on cell therapy for cardiology application.

Many clinical studies of stem cell therapy in cardiology make use of BMC samples [4]. Issues related to molecular mechanisms and their relevance to the efficiency of cell therapy for heart repair have been rarely investigated. Considering the importance of stem cell homing to the cardiomyoplasty outcome [6], this study analyzed its activation status through the expression of key molecules involved in this mechanism, the chemokine SDF-1 and its receptor CXCR4 in BM samples of patients with ischemic and valvular heart diseases. The activation of this molecular axis is primordial to the start of the tissue repair process, such as what occurs after myocardial injury [11].

The activation and release of chemokines is a key mechanism in response to myocardial injury. In addition, there are multiple factors involved in homing of stem cells to heart diseases, which may in turn affect the production of chemokines in these conditions [12]. Among these, specific mention can be made to growth factors, as stem cell factor (SCF), and vascular endothelial growth factor (VEGF); cell adhesion molecules, as integrins, intercellular adhesion molecule-1 (ICAM-1), Monocyte Chemoattractant Protein-1 (MCP-1); and nitric oxide. SDF-1, particularly the α isoform, attracts stem cell to the lesion areas [7, 8], playing an essential role in protecting and repairing the ischemic organ. The increase of SDF-1 expression in response to a hypoxic environment [13], such as ischemia, acts as a cellular signal to attract CXCR4⁺ stem cells, which are potentially beneficial to cardiac repair [7]. Here, we evaluated plasma levels of SDF-1 in the BM of patients; and the data in literature reporting expression levels of this chemokine by BM are limited. However, even though showing reduction in cardiac patients, the chemokine levels did not differ significantly from healthy controls. Studies have shown controversial data regarding the levels of SDF-1 in pathological and physiological situations. Wyderka et al. showed decreased systemic levels of SDF-1 in ischemic patients when compared to those with valvular disease [14]. Chang et al. observed that serum levels of SDF-1 did not differ between AMI patients (1304 ± 591 pg/mL) and healthy control subjects (1264 ± 250 pg/mL) [15]. However, a recent study showed that patients with acute coronary syndrome (ACS) have high levels of systemic SDF-1 when compared to healthy control subjects. Furthermore, the authors identified SDF-1 as a potential biomarker for CVD in this cohort of patients [16]. Another key factor is the context of SDF-1 modulation, whether acute or chronic. Researchers found that the regulation of this chemokine is transient, with a significant increase in the acute phase and consequent decrease in the chronic phase of ischemia [17, 18]. This decline is due to the inactivation of SDF-1 by proteolytic enzymes and metalloproteinases [19].

Moreover, we could not evaluate the relationship between circulating and medullary concentrations of SDF-1. However, we believe that the SDF-1 values found in this study reflect the bioavailability of this molecule for tissue repair and regeneration, promoting the homing of circulating CXCR4⁺ cells. Inflammatory mediators, released in response to ischemic injury to limit tissue damage and create conditions for the start of the repair [12], may also have an effect on SDF-1 expression [20]. Here, we showed a trend in association between SDF-1 and IL-6 in IHD patients; both were slightly decreased in relation to the VHD group. Moreover, the levels of TNF-α were quite reduced in these patients. Thus, it is possible that, even within the BM, these molecules are necessary to trigger the homing, a process that may have been harmed in the ischemic patients.

Additionally, in our study, SDF-1 levels were decreased in VHD patients undergoing pharmacological therapy with beta-blockers compared to those who did not use medication. Zou et al. (2012) suggested that this pharmacologic class interferes in stem cell homing by decreasing SDF-1 and CXCR4 expression [21]. In fact, the IHD patients made more use of these drugs than the VHD and this may also be speculated as preventing the activation of homing.

Most of part of the biological effects of the chemokine SDF-1, including cardiac repair, is mediated by CXCR4/CD185 receptor [8]. For the interaction to occur, the receptor needs to be functionally active at the cell surface and coupled to G-protein [22]. Another receptor with high affinity to SDF-1 has been identified, the CXCR7 receptor. Studies suggest that CXCR7 perform as a supporting or receptor modulator to CXCR4 signaling and also be involved in the regulation of processes mediated by SDF-1 [9].

Evidence indicates that the levels of expression of CXCR4 on BMC surface determine the efficiency of homing [23]. In our study, there was no difference in the number of CXCR4⁺ BMC between the groups. By evaluating differences in the magnitude of CXCR4 and CXCR7 gene expression, we found high levels of mRNA of both receptors in the VHD group. Analyzing the expression of CXCR4 and CXCR7 to each patient individually, not necessarily the expression of two receivers was similar. Interestingly, we found that CXCR7 was higher for many patients, where the CXCR4 was low. Moreover, the increased CXCR4 mRNA expression was not accompanied by an increase in protein expression on the BMC surface. It should be observed that changes in mRNA expression levels do not necessarily reflect changes in protein expression levels, and therefore, it is important to determine the expression of the functional protein, which was a limitation of our study. Previous studies demonstrated that the expression of CXCR4 on the surface of the membrane is unstable, independent of the regulation of transcription, and effectively regulated by internalization receptor [24]. Thus, the intracellular storage of CXCR4 may at least partly explain the fact that increased mRNA expression of this receptor in the VHD group does not lead to an increase in CXCR4⁺ BMC.

Damas et al. observed in patients with angina, low levels of CXCR4 on the surface of peripheral blood mononuclear cells despite increased expression of the corresponding gene, as evidenced by mRNA levels in these cells [25]. In addition, the chronic phase of IHD may be related to low levels of gene expression of CXCR4 in this group of patients. Van Weel et al. have verified a decrease in mRNA expression of CXCR4 in the chronic phase of ischemia compared with the acute phase in patients with peripheral artery disease [26]. Thus, according to our data, factors such as SDF-1 levels, pharmacological treatment, and expression on the membrane surface may affect the expression and functionality of CXCR4, and consequently functional response of BMC. Efforts to mobilize the internalized receptor and increase the CXCR4⁺ cell expression [13, 27] may be of great value to reinforce the benefits of cell therapy for heart repair.

Despite a small sample size, we could demonstrate the expression profile of SDF-1, CXCR4 in a considerable number of cardiac patients, which allows us to infer the cell homing activation status of BMC in these patients. Considering that the chemoattractant function of SDF-1 correlates with the expression of CXCR4 on the cell surface [7], we found a strong direct correlation between CXCR4⁺ cells expression and SDF-1 plasma levels in the VHD group. This result is supported by the fact that these patients have shown higher SDF-1 levels as compared with the ischemic group. Then, one may assume that there may be a minimum level of the chemokine necessary to stimulate the activation of CXCR4⁺ cells in the BM. Additionally, it is worth mentioning the difference between bone marrow sources used in the study, since the control group samples obtained from the iliac crest, therefore for ethical reasons, it is unfeasible to collect MO sternum of healthy subjects, which is a limitation of the study. Issues such as the origin, regulation and impact of SDF-1 in circumstances that require tissue regeneration still remain incompletely understood. Finally, due to the difficulty in obtaining bone marrow samples not all study participants were evaluated for the expression profile of all molecules, a major limitation of this study.

Regarding the CXCR7 receptor, to date little is known about the function of this receptor during myocardial disease. In this study, it was observed a failure on the regulation of CXCR7 and CXCR4 receptors in both IHD and VHD groups. Some authors have hypothesized that the CXCR7 acts as a non-signaling receptor by removing extracellular SDF-1, and consequently, indirectly controlling the signaling of CXCR4 [28]. Studies reported that both the population of cells analyzed and the context (health/disease) may be considered contributing factors for the CXCR7 expression profile [29]. However, in view of the limitation of obtaining biological material of patients, we could not evaluate the expression of this receptor in the population of bone marrow cells evaluated in the study.

Our data demonstrate that the expression profile of key molecules involved in cell homing was altered in patients with heart disease. According to this profile and the central role of SDF-1/CXCR4 signaling in cell homing, our data show a trend towards decreased activation of this pathway in patients with IHD. Such functional impairment of BMC in cardiac patients may limit their therapeutic potential for clinical cell therapy. Factors such as pharmacotherapy, chronic phase of the disease, among others, could compromise the expression levels of these molecules, and hence the capacity to undergo cell therapy. The standard drug therapy in cardiology, especially with beta-blockers, deserves special attention because it can interfere with the expression of molecules involved in homing, jeopardizing this mechanism, a fact that should be considered in clinical trials on cell therapy, particularly for clinical application in cardiology. However, more studies are needed to establish the activation status of cell homing in the heart diseases, and the expression levels of molecules involved in this mechanism for therapeutic purposes.

Pathophysiological differences between IHD and VHD can affect the molecules involved in the activation of homing. Here, expression the key molecules involved in activation of homing, as the chemokine SDF-1 and its receptor CXCR4 and CXCR7, showed variations once analyzed in BM samples of patients with ischemic and valvular heart diseases. We could verify that both cardiovascular diseases differentially affect the levels of these molecules, as well as, the inflammatory factors. In addition, the use of beta-blockers appears to interfere in this mechanism, a fact that should be considered in protocols that use BMC.