Abstract
Background: Elevated mortality rates in patients with metabolic syndrome (MetS) are partly due to adverse remodeling of multiple organs, which may lead to cardiovascular disease, nonalcoholic fatty liver disease, kidney failure, or other conditions. MetS symptoms, such as obesity, hypertension, hyperglycemia, dyslipidemia, associated with insulin and leptin resistance, are recognized as major cardiovascular risk factors that adversely affect the heart. Summary: Pathological cardiac remodeling is accompanied by endothelial cell dysfunction which may result in diminished coronary flow, dysregulated oxygen demand/supply balance, as well as vessel rarefaction. The reduced number of vessels and delayed or inhibited formation of collaterals after myocardial infarction in MetS heart may be due to unfavorable changes in endothelial cell metabolism but also to altered expression of vascular endothelial growth factor molecules, their receptors, and changes in signal transduction from the cell membrane, which severely affect angiogenesis. Key Messages: Given the established role of cardiac vessel endothelial cells in maintaining tissue homeostasis, defining the molecular background underlying vessel dysfunction associated with impaired angiogenesis is of great importance for future therapeutic purposes. Therefore, the aim of this paper was to present current information regarding vascular endothelial growth factor signaling in the myocardium of MetS individuals.
Introduction
The high mortality in patients with metabolic syndrome (MetS) is caused mainly by multiorgan dysfunction, as a result of increased cardiovascular risk factors, such as hypertension, obesity, and diabetes [1]. In combination, factors such as abdominal obesity, elevated blood pressure, an altered lipid profile, and abnormal carbohydrate metabolism play a crucial role in the pathogenesis of MetS [2]. MetS symptoms are mostly a result of poor lifestyle choices (smoking, unbalanced diet, and inadequate physical activity) and severely affect the EC metabolism due to oxidative stress and activation of pro-inflammatory pathways [3]. Structural remodeling of cardiac vessels including changes in extracellular matrix collagen-elastin ratio and vascular smooth muscle proliferation, but also impaired vasomotor function due to alterations in mineralocorticoid signaling, change mechanical properties of vessels, and affect blood flow. This, in turn, negatively affects oxygen and nutrient demand/supply balance [4, 5]. Impaired cardiac blood flow prompts adverse cardiac tissue remodeling which results in the development of one of the main MetS pathologies – heart failure with preserved ejection fraction [6, 7].
MetS microenvironment may also diminish the ability of ECs to respond to angiogenic stimuli [3]. In metabolically healthy animals, therapeutic proangiogenic agents, such as vascular endothelial growth factor (VEGF), stimulate collateral growth in cardiac muscle after induced heart infarction. These agents may reduce the size of the infarct and the risk of death. However, similar trials in patients with coronary disease have mostly failed [8, 9]. A diminished response of ECs to angiogenic stimuli is probably a result of their changed metabolism, reduced NO bioavailability, and elevated oxidative stress [8, 10]. Another cause of delayed or inhibited collateral growth in individuals with MetS may be a delayed phenotypic switch of vascular smooth muscle cells (VSMCs). During collateral vessel formation, VSMCs undergo the change from a contractile toward a proliferative phenotype, but to sustain blood vessel formation, they need to return to the contractile phenotype, which is inhibited in MetS [11]. Finally, MetS is associated with inflammation, which results in elevated expression of adhesion molecules on EC surfaces and inadequate infiltration by inflammatory cells, such as neutrophils, which in turn may affect collateral growth in MetS myocardium [12]. A poor angiogenic response and the accompanying EC death may, in turn, lead to a diminished density of micro-vessels and arterioles in cardiac muscle [4, 13‒17].
The term “angiogenesis” describes the growth of new vessels via sprouting from preexisting ones. Under the influence of proangiogenic factors, ECs become motile and change their shape to form tip cells (elongated cells that become the spearhead of the sprout) and stalk cells (which establish the new vessel lumen and elongate the sprout via rapid proliferation). Finally, the new structure recruits mural cells, which stabilize it to enable blood flow (extensively reviewed in [18, 19]). Angiogenesis is regulated by several groups of different proteins. The most important ones include VEGFs, their receptors and coreceptors, as well as downstream cytoplasmic signaling molecules [20]. The VEGF family consists of five members – VEGF-A, VEGF-B, VEGF-C, VEGF-D, and the placental growth factor, which act via three basic receptors – VEGFR1, VEGFR2, and VEGFR3. Both the growth factors and receptors may occur in proangiogenic and antiangiogenic isoforms, which enables subtle and precise regulation of angiogenesis (extensively reviewed in [21]). Not only VEGF molecules and their receptors play an important role in angiogenesis. The proper functioning of downstream signaling pathways is equally important [22]. Clinical observations show increased serum VEGF levels in patients with MetS [23]. Thus, levels of VEGF positively correlate with body mass index, obesity, and the amount of visceral adipose tissue [24, 25]. Serum VEGF elevation has been reported in type 2 diabetes mellitus, but only in patients with vascular complications [26]. On the other hand, VEGF levels are normal when blood glucose concentration is controlled [27]. Individuals with hypertension also have higher serum VEGF levels than normotensive individuals [28]. Despite higher VEGF levels, blood vessel formation and collateral vessel growth in patients with MetS are impaired after myocardial infarction [9]. This may be a result of EC dysfunction but also of the adversely affected VEGF signaling cascade.
Therefore, the aim of this paper was to present current information regarding VEGF signaling pathways, with a particular emphasis on VEGF-A and VEGFR2 in ECs in cardiac tissue during obesity and MetS. We searched through the PubMed database using key words such as “VEGFR1”, “VEGFR2”, “PLC-γ-ERK1/2”, “SRC”, “AKT”, “MAPK”, “VEGF signaling”, “cardiac vessel”, “heart failure”, and “metabolic syndrome” and paid particular attention to literature reports from the last 5 years.
Vascular Endothelial Cell Growth Factors and Their Cellular Receptors
Molecules from the VEGF family have an affinity for transmembrane-spanning receptors that contain an intracellular domain with tyrosine kinase activity and seven immunoglobulin-like extracellular domains. These domains are present in three isoforms of VEGF receptors – VEGFR1, VEGFR2, and VEGFR3 [20]. VEGFR1 (Fms-like TK-1, Flt-1) was described and characterized in the early 1990s [29]. This receptor binds with the greatest affinity to VEGF-A and regulates its biological effects. Although VEGFR1 strongly binds VEGF-A, it has a much weaker kinase activity when compared with that of VEGFR2. Therefore, the biological effect of VEGF-A via VEGFR1 activation is much weaker than that exerted via VEGFR2. VEGF-A/VEGFR1 signaling cascade is not crucial for angiogenesis initiation but for organization of new vessels maturation during later stages of this process [30, 31]. Since there are also different VEGF-A isoforms, VEGFR1 can form homodimers or heterodimers with VEGFR2, depending on the type of ligand. The heterodimers mainly limit VEGF-A-regulated activation of the receptor [32]. As a result of alternative splicing, a soluble, truncated form of VEGFR1 may be also formed [33]. Soluble VEGFR1 binds to VEGF-A and limits its biological availability, thus inhibiting VEGF function [33, 34]. Furthermore, by binding to low-density lipoproteins (LDL) and subsequent receptor phosphorylation, endocytosis, and degradation, VEGFR1 plays a crucial role in regulating the absorption of fatty acids by ECs [35].
The VEGFR2 subtype is another protein involved in VEGF/VEGFR signaling [36]. It plays an important role in early angiogenesis/vasculogenesis. Activation of the receptor occurs after binding a VEGF molecule, which causes VEGFR2 dimerization [37]. LDL may also activate this receptor [38]. In a most common pathway, the receptor is activated by VEGF-A but also by VEGF-C and VEGF-D [39]. Major coreceptors in VEGFR2 signaling are neuropilins (NRP). NRP-1 is mainly expressed in arterial ECs and binds VEGF-A, especially VEGF-A165. NRP-1 binds with VEGFR2 which results in an up to six times greater activation of this heterodimer when compared to activation without coreceptor. NRP-2 binds rather VEGF-C and enhances the effect of VEGFR3 activation [40]. VEGFR2 can form heterodimers with two other receptor subtypes – VEGFR1 and VEGFR2, which modulate VEGF signaling. VEGFR2 interacts with VEGFR1, which may sensitize ECs to VEGF, since VEGFR2 is phosphorylated by a VEGFR1 tyrosine kinase domain after heterodimerization. Finally, as a result of alternative splicing, also soluble form of VEGFR2 can be produced, which performs similar functions as soluble VEGFR1, including angiogenesis inhibition [20, 41].
VEGFR3, which is also known as Flt-4, is crucial for lymphatic vessel growth [42]. Moreover, new blood vessel formation is also dependent on VEGFR3 expression in blood endothelial cells [43]. Interaction of VEGFR3 with VEGFR2 results in the formation of a VEGFR2/VEGFR3 heterodimer, which regulates initial steps of angiogenesis [39]. Integrin-β1, syndecan-4, and neuropilin-2 (NRP-2) help VEGFR3 function normally [44].
Downstream Signaling in VEGF/VEGFR Pathways
VEGF receptor activation activates secondary messengers in cell cytoplasm. The most important molecules that are involved in VEGF signaling are phospholipase C γ (PLC-γ)/extracellular signal-regulated kinase (ERK1/2), phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, AKT)/mammalian target of rapamycin (mTOR), and SRC kinase pathways. In addition, activation of the VEGFR2 receptor triggers p38 mitogen-activated protein kinase (p38 MAPK) and signal transducer and activator of transcription (STAT) proteins [20].
The PLC-γ-ERK1/2 pathway plays a crucial role in postnatal angiogenesis. Dimerized VEGFR2 receptor undergoes phosphorylation at Y1173 in rodents and Y1175 in humans, which indirectly activates ERK1/2 and/or the nuclear factor of activated T cells (NFAT) [45]. First, PLC-γ is activated, which initiates production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). As a result of IP3 action, calcium ions are released from the endoplasmic reticulum and activate calmodulin and a serine-threonine kinase, e.g., calcineurin. These changes in cell signaling decrease VEGFR1 expression and enhance VEGFR2 proangiogenic function [20, 46]. DAG pathway activates protein kinase C (PKC) and then ERK1/2, which regulates ECs specification, proliferation, and migration by activating the E26 family of transcription factors (ETS) or causing the phosphorylation of histone type 7 deacetylases (HDAC7) [47].
The AKT serine-threonine kinase pathway regulates many processes in ECs. AKT is involved in the regulation of cell survival, motility, and vascular permeability [48]. Activation of this kinase requires binding through its PH domain with phosphatidylinositol-3,4,5-trisphosphate (PIP3), which is produced from phosphatidylinositol-4,5-diphosphate (PIP2) by a phosphoinositide 3-kinase (PI3K). The AKT-1 kinase, which works through the mTOR2 complex, is the most important molecule involved in the regulation of angiogenesis. VEGFR2 does not contain a domain, which can bind directly with PI3K, so activation of this kinase is mediated either by protein-tyrosine kinase SRC, VE-cadherin receptor domain, or AXL receptor tyrosine kinase (AXL) [20].
The SRC-dependent pathway is crucial for changes in cell shape, cell migration, and cell polarization, as well as regulating vascular permeability by intercellular junction modulation, e.g., via VE-cadherin phosphorylation in response to VEGF. SRC activation is determined by VEGFR2 phosphorylation at Y949 in rodents and Y951 in humans. Activated receptor binds T-cell-specific adapter (TSAd), which activates the SH3 domain of SRC. Moreover, shear stress accelerates SRC activation and controls the interaction between the cytoskeleton and cell adhesion proteins [49]. The SRC kinase also affects micro-vessel vessel permeability, phosphorylating VE-cadherin that results in the endocytosis of this protein and the destruction of adherent junctions, which disrupts vascular barrier [50].
VEGF signaling pathways can also involve p38 MAPK and STATs. MAPK induces angiogenesis, mostly by stimulating cell migration and EC survival. This pathway can also affect vascular wall permeability. Activation of p38 MAPK is dependent on NRP-1 costimulation of VEGFRs and the entry of calcium ions from the endoplasmic reticulum into cell cytoplasm. Ca2+ influx activates protein kinase 2β (PTK-2β), which – together with SRC – triggers the p38 MAPK cascade [20]. In addition, the STAT-1 and STAT-3 proteins are involved in cell cycle regulation, apoptosis, and activation of inflammatory molecules in the endothelium. STAT-3 also increases the density of vessels [51]. Main VEGF-VEGFR signaling pathways are summarized in Figure 1a.
VEGF-A-VEGFR2 signaling cascade in normal (a) and MetS/obesity (b) conditions. a Main signaling molecules involved in signal transmission via the VEGF-A-VEGFR2 and VE-cadherin axis. b MetS/obesity conditions affect the VEGF-A/VEGFR2/VE-cadherin signaling axis: (1) reduce AKT pathway activity and upregulate the PLC-γ pathway; (2) reduce the expression of VE-cadherin and elevate the internalization of these molecules.
VEGF-A-VEGFR2 signaling cascade in normal (a) and MetS/obesity (b) conditions. a Main signaling molecules involved in signal transmission via the VEGF-A-VEGFR2 and VE-cadherin axis. b MetS/obesity conditions affect the VEGF-A/VEGFR2/VE-cadherin signaling axis: (1) reduce AKT pathway activity and upregulate the PLC-γ pathway; (2) reduce the expression of VE-cadherin and elevate the internalization of these molecules.
Alterations in VEGFRs Expression in MetS
Despite a higher serum concentration of VEGFR ligands, individuals with MetS do not seem to have increased angiogenesis. One suspected mechanism behind this phenomenon is associated with altered VEGFR expression and increased levels of soluble VEGF receptors, which decrease VEGF bioavailability [41]. In the myocardium of db/db mice, known to develop MetS symptoms, a decrease in the expression of both VEGFR1 and VEGFR2 receptor subtypes is observed [52]. In the myocardium of obese and insulin-resistant rats, the expression of VEGFR1 and VEGFR2 mRNAs is significantly decreased. Furthermore, the expression of PECAM-1 protein, which is a molecular marker of endothelium, is also impaired in fa/fa diabetic rats, simultaneously with a diminished collateral vessel formation in these rats [53]. A reduction in microvascular and arteriolar density was also observed by Zeng et al. in db/db mouse hearts [14].
The presence of soluble VEGFR forms, which are responsible for limiting ligand bioavailability, is extremely important in VEGF signaling pathways. There is a significant increase in the amount of soluble form of VEGFR2 in the serum of patients with MetS symptoms, whereas there is no change in the concentration of soluble VEGFR1 [41]. On the other hand, hyperlipidemia, one of MetS symptoms, may increase the expression of VEGFR1, which has negative effects on angiogenesis and vessel integrity, as it was observed in zebrafish and mouse models [54]. Despite VEGF overexpression, there may be a poor angiogenic response of ECs in the heart in MetS, which is related to the altered VEGF signaling pathway. This poor angiogenic response can result from at least two mechanisms: (1) reduced VEGFR expression and/or (2) elevated concentration of sVERFR-2, which leads to limited VEGF-A availability.
VEGFR2 Downstream Signaling Cascade Alterations in MetS
PLC-γ/ERK1/2
There are limited data regarding the details of the PLC-γ-ERK1/2 pathway in the heart over the course of MetS. The existing data show that there are no major changes in the expression of ERK pathway-dependent kinases in the cardiomyocytes of db/db mice with MetS [55]. On the other hand, an increased activation of ERK in the whole myocardium observed in db/db mice may suggest that this pathway may be affected in ECs or other non-cardiomyocyte cardiac cells [56]. In the hearts of Zucker diabetic fatty (ZDF) rats, ERK kinase phosphorylation is increased, indicating ERK kinase activation [57]. These observations suggest that ECs can be a potential source of the observed changes in ERK signaling in the myocardium, since ECs are more numerous than either fibroblasts or macrophages. Moreover, an increased ERK1/2 phosphorylation (and thus ERK1/2 activation) has been observed in the aorta of diet-induced obese and diabetic rats. Of interest, physical exercise in these animals reduced ERK1/2 activation [58]. Conversely, ERK phosphorylation was not detected in a porcine model of MetS [59]. Changes in lipid metabolism, which also occur in MetS, can impact ERK signaling. The addition of the very low-density lipoproteins (VLDLs) isolated from MetS individuals and added to atrial myocyte cell lines cultured in vitro resulted in a diminished dephosphorylating activity of calcineurin and an increased cytoplasmic concentration of phosphorylated NFAT; additionally, the amount of activated NFAT was reduced in the cell nucleus, which results in alteration of myofilament protein expression, disruption of sarcomere, and atrial myopathy [60]. Based on the scarce data regarding the VEGFR signaling in the heart in MetS, the ERK pathway is mostly upregulated, but further studies in this field are needed to assess the role of ERK activation, especially in the cardiac endothelium.
PI3K/AKT
In the hearts of db/db mice with MetS, a decrease in the concentration of active AKT was noted [52]. No difference in the AKT activity was observed, when only cardiomyocytes were analyzed, which suggests that this pathway may be affected in other cell types, such as fibroblasts or ECs [55]. Similarly, AKT phosphorylation is reduced in the hearts of ZDF rats, known to present MetS symptoms [57]. Diminished AKT phosphorylation was also confirmed in a diet-induced rat MetS model [61]. High-fructose diet results in the development of MetS symptoms and reduces AKT activity [62]. In another animal model (JCR:LA-cp rats), AKT activation failed in the heart during repetitive ischemia, which may explain diminished collateral formation; the observed phenomenon was linked to an altered REDOX state in MetS heart since when the levels of ROS were controlled, also the activity of AKT was restored [63]. Furthermore, in a high-cholesterol diet swine model, reduced cardiac phosphorylation of AKT was observed [64]. Some contradictory data are also available. In a porcine model of myocardial infarction, the cardiac AKT signaling pathway was activated in MetS, unlike that in the control group [59]. To sum up, the AKT signaling pathway in MetS is presumably downregulated or even blocked, which may exert a detrimental effect on the angiogenic response of ECs.
Src/VE-Cadherin
VE-cadherin phosphorylation, which is regulated via VEGF/VEGFR2/Src axis, disrupts EC adherens junctions and promotes vascular leakage [65]. On the other hand, destabilization of adherens junctions is an essential initial step of EC migration, necessary for new vessel formation [66]. Additionally, VE-cadherin may interact with VEGFR2 to prolong these receptors’ half-life, and it has a positive impact on EC survival [67]. In a high-fat diet (HFD) porcine model, VE-cadherin expression is reduced after myocardial infarction, which indicates angiogenic pathway disruption and may not only promote vascular leakage but also diminish the proangiogenic response of ECs [68]. Of note, downregulation of VE-cadherin expression may be also caused by elevated numbers of neutrophils in cardiac tissue, as was described in a HFD mouse model [69]. Neutrophil infiltration in MetS may negatively affect collateral growth in MetS myocardium; one of the possible mechanisms may be an extensive release of neutrophil elastase that promotes VE-cadherin degradation, inhibits VE-cadherin expression, and causes vascular leakage [12, 69].
Src/p38 MAPK/AKT
Src, p38 MAPK, and AKT pathway activation, crucial for collateral growth in cardiac tissue, is redox dependent, and since ROS levels are elevated in MetS, an abnormal activation of above molecules may, at least in part, be responsible for compromised new vessel formation [63, 70, 71]. In ZDF rats and in db/db mice, phosphorylation of p38 MAPK was elevated in obese and diabetic animals. In this experiment, examination was performed on the whole cardiac tissue. Since the sample consisted mostly of cardiomyocytes and elevated p38 MAPK activity may correlate with inflammatory processes, these data probably do not describe events occurring in ECs [56, 57]. Activation of the p38 MAPK pathway in the aorta and coronary arteries was increased in MetS swine [64]. Summarizing, MAPK phosphorylation and thus activation is probably reduced in MetS hearts, although data are contradictory. Activation of p38 MAPK is strictly connected with REDOX state of the ECs, and an angiogenic response to VEGF is only possible when ROS levels are controlled. Altered activation of transcription factors regulated by phosphorylated MAPK proteins was also observed; for example, STAT-3 activation is reduced in the cardiac tissue of db/db mice [56]. Main VEGF-VEGFR signaling pathway alterations are summarized in Figure 1b.
Conclusion
There are a growing number of individuals with MetS worldwide. These patients develop HF, whose exact pathophysiology, especially in terms of vessel/capillary rarefaction associated with insufficient angiogenesis, is not fully understood. Moreover, it becomes increasingly problematic to diagnose and treat this complex disease that generates huge socioeconomic costs and markedly impairs health-related quality of life. MetS etiology and symptom progression leading to HF seem to be associated with adverse alterations in multiple metabolic pathways, leading to disrupted EC homeostasis (EC dysfunction) and impaired angiogenesis, followed by micro-vessel regression and collateral growth delay, all of which are recognized as early unfavorable pathological remodeling of cardiac muscle during MetS. Dysfunction of receptors for various VEGF molecules and their secondary messengers is partially responsible for impaired angiogenesis, reduced blood capillary density, and increased microvascular permeability in the hearts of individuals suffering from MetS symptoms. Poor angiogenesis mediated by the VEGF/VEGFR axis is largely unknown in MetS and should be further investigated. Elucidation of the precise mechanisms regulating angiogenesis in MetS would lead to a better understanding of the mechanisms behind MetS-induced cardiac dysfunction and would be a stepping stone in the search for novel therapies of this clinical condition.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
This article has received funding from the Ministry of Science and Higher Education, Grant No. D/2018020048, “Diamond” Grant (to K.B.). The funder had no role in the design, data collection, data analysis, and reporting of this study.
Author Contributions
K.B., M.B., E.J.-S., A.R., M.K., O.A., and J.N.-B.: literature analysis and final approval of the version to be published; K.B. and J.N.-B.: conception of design and drafting the work; and E.J.-S., A.R., M.K., and O.A.: reviewing the work critically for important intellectual content.