Epidermal growth factors in the kidney and relationship to hypertension

Abstract

Members of the epidermal growth factor (EGF)-family bind to ErbB (EGFR)-family receptors that play an important role in the regulation of various fundamental cell processes in many organs including the kidney. In this field, most of the research efforts are focused on the role of EGF-ErbB axis in cancer biology. However, many studies indicate that abnormal ErbB-mediated signaling pathways are critical in the development of renal and cardiovascular pathologies. The kidney is a major site of the EGF-family ligands synthesis, and it has been shown to express all four members of the ErbB receptor family. The study of kidney disease regulation by ErbB receptor ligands has expanded considerably in recent years. In vitro and in vivo studies have provided direct evidence of the role of ErbB signaling in the kidney. Recent advances in the understanding of how the proteins in the EGF-family regulate sodium transport and development of hypertension are specifically discussed here. Collectively, these results suggest that EGF-ErbB signaling pathways could be major determinants in the progress of renal lesions, including its effects on the regulation of sodium reabsorption in collecting ducts.

members of the epidermal growth factor (EGF) family are small mitogenic proteins involved in a number of mechanisms such as normal cell growth and differentiation. Their effects are mediated via autocrine, paracrine, or endocrine mechanisms. ErbB receptors have been recognized as targets in anticancer therapy and are now used in the treatment of breast and colon malignancies (93). The name and gene symbol ErbB (Table 1) was derived from a viral oncogene to which these receptors are homologous: erythroblastic leukemia viral oncogene. Multiple epithelial cancers, including kidney carcinoma, are associated with the EGFR-EGF axis (61, 73, 111, 134). In addition to cancer biology, EGF receptor (EGFR) activation is critical in acute kidney injury (AKI) and chronic kidney diseases (CKD; Refs. 71, 108, 130). Here we discuss recent results supporting functional interactions between EGF-family proteins and blood pressure control in the kidney, as well as provide details of some mechanisms underpinning such interactions and the clinical consequences of these modulations. Particular emphasis is placed onto the mechanisms of regulation of sodium transport in the kidney by the EGF family growth factors. Hence we aim to provide critical insight into the physiological control of kidney function by EGF and its related growth factors.

Table 1.

Gene/chromosome locations of ErbBs

Protein	Gene/Chromosome Location (Human)	Gene/Chromosome Location (Mouse)	Gene/Chromosome Location (Rat)
EGFR (ErbB1/HER-1)	Chr 7	Chr 11	Chr 14
	55,086,725–55,275,031	16,652,206–16,813,910	97,617,358–97,788,211
ErbB2 (neu/HER-2)	Chr 17	Chr 11	Chr 10
	37,844,393–37,884,915	98,273,798–98,299,030	87,219,157–87,242,919
ErbB3 (HER-3)	Chr 12	Chr 10	Chr 7
	56,473,809–56,497,291	128,006,424–128,026,557	1,858,057–1,877,353
ErbB4 (HER-4)	Chr 2	Chr 1	Chr 9
	212,240,442–213,403,352	68,086,540–69,154,633	66,843,898–67,967,970

Medical College of Wisconsin Rat Genome Database website (http://rgd.mcw.edu/) was used as a source for gene/chromosome locations.

EGFR, epidermal growth factor receptor.

ErbB Receptors

The EGFR family, also known as ErbB receptors (91), consists of four transmembrane receptors that belong to the receptor tyrosine kinase superfamily and includes EGFR (ErbB1/HER-1), ErbB2 (neu/HER-2), ErbB3 (HER-3), and ErbB4 (HER-4). EGFR as well as other receptors in this family is a transmembrane receptor that consists of an extracellular domain with conserved two cysteine-rich domains necessary for ligand binding; a single membrane-spanning domain, which has a passive role in signaling and functions as an “anchor” of receptor in plasma membrane (53), and a cytoplasmic protein tyrosine kinase domain, where six tyrosine autophosphorylation sites are located (Fig. 1). After ligand binding, ErbB receptors dimerize, which is a required critical step for intrinsic receptor tyrosine kinases to be activated and thereby specific tyrosine-containing residues to become autophosphorylated (12). Phosphorylated ErbB receptors can recruit different adapter proteins with Src homology domain-2 (SH2) or phosphotyrosine binding domains (PTB; Ref. 8). Signals from dimerized, activated ErbB receptors lead to activation of multiple intracellular signal transduction pathways. Depending on the pairing of ErbB family receptors (homodimers or heterodimers; Fig. 1), there can be downstream stimulation of different combinations of signaling pathways (43).

Fig. 1.

A schematic representation of ErbB receptors and their ligands. ErbB receptor is a transmembrane receptor that consists of an extracellular ligand binding domain, a transmembrane region, and an intracellular domain that includes an inactive (in the absence of ligand) tyrosine kinase region. Binding of a specific growth factor to ErbB transforms its extracellular domain into a configuration that enables formation of a homodimer or a heterodimer and activates the intracellular kinase domain. TGF-α, transforming growth factor-α. EGFR, epidermal growth factor receptor; HB-EGF, heparin binding-epidermal growth factor.

To some extent, EGFR is expressed in every nephron segment. Although EGFR is the predominant ErbB receptor found in the normal adult mammalian kidney tubule, ErbB2, ErbB3, and ErbB4 are also expressed in the kidney but mainly localize to the distal and connecting tubules and collecting ducts (87, 130, 131, 133). Figure 2 demonstrates representative immunohistochemical staining for ErbB2 receptor in the kidney cortex of Sprague-Dawley rat. A number of various mechanisms control ErbB receptors trafficking and degradation (64, 99) as well as polarized distribution (18, 127) in the renal epithelial cells.

Fig. 2.

Immunohistochemical staining of ErbB2 receptor distribution in the kidney cortex. Representative images of kidney sections of Sprague-Dawley rats are shown at ×20 and ×40 magnifications. ErbB2 is predominantly distributed at basolateral surface of the collecting ducts.

ErbB Ligands

Regulation of ErbB receptors function is controlled by their ligands, members of the EGF-related peptide growth factor family (39). All EGF-family members, like many other growth factors, are derived from membrane-bound precursor proteins (97, 128). There are at least 12 ligands identified so far that can bind ErbBs and induce dimerization of distinct functional receptor pairs. EGF, transforming growth factor-α (TGF-α), and amphiregulin are specific for EGFR and primarily induce the formation of EGFR/EGFR homodimers and EGFR/ErbB2 heterodimers (27). Heparin binding-EGF (HB-EGF), betacellulin, and epiregulin can bind to either EGFR or ErbB4. Neuregulins-1 and −2 can bind both ErbB3 and ErbB4; neuregulins-3 and -4 are specific only for ErbB4 (71). Currently, no ligands have been identified for ErbB2 homodimers. However, ErbB2 is the preferred heterodimerization partner of other ErbB family members and ErbB2-containing heterodimers have the strongest signaling output (36, 114). Interestingly, the sequence of the ErbB3 catalytic domain suggests that this receptor does not have receptor tyrosine kinase activity (17). Thus ErbB3 may function as a platform for heterodimerization and subsequent transphosphorylation by other members of the ErbB family. Hence ErbB2 and ErbB3 can be activated through heterodimerization (6, 17).

Most of the ErbB-family ligands are expressed in the kidney (see Table 2 for some examples), and their expression could be dramatically changed during renal development or in pathological conditions (49, 71, 130). EGF and its related growth factors are present in the lumen at levels much higher than in plasma. However, ErbB receptors are expressed at the basolateral surfaces of tubules in normal adult kidneys (see Fig. 2). This may serve as a protective mechanism for the distal nephron, which can be disturbed under some pathophysiological conditions. One of the examples of apicobasal polarity abnormalities is mislocalization of ErbB receptors that is observed in a variety of genotypic and phenotypic animal models as well as in humans with polycystic kidney disease (PKD; Ref. 127). Another potential mechanism that emerges under some pathological conditions is a disturbance of epithelial cellular integrity. Following tubular injury, tight junctions can be disrupted and ErbB receptor ligands can traverse to the basolateral surface and consequently activate corresponding receptors.

Table 2.

EGF family growth factor expression in the kidney

Protein	Expression Reported	References
EGF	TAL, DCT, CNT, CDs, papilla	(11, 30, 34, 49, 79, 81, 89, 118)
TGF-α	TAL, DCT, CDs	(11, 35, 68, 79, 117)
HB-EGF	Podocytes, mesangial cells, parietal epithelial cells, DCT, PCT, CDs	(10, 29, 44, 68, 74, 77, 78, 88, 98, 116)
Amphiregulin	PCT, CDs, podocytes, mesangial cells	(58, 68, 74)
Epiregulin	mesangial cells	(74)

TGF-α, transforming growth factor-α; HB-EGF, heparin binding-epidermal growth factor; PCT, proximal convoluted tubule; TAL, thick ascending limb of Henle's loop; DCT, distal convoluted tubule; CNT, connecting tubule; CDs, collecting ducts.

Activation of ErbB Receptors and Consequent Physiological Functions

As ErbB ligands exist as inactive transmembrane precursors, they need proteolytic cleavage of their ectodomain to be released as mature soluble ligands. All EGF-family members are synthesized as membrane-anchored precursors that can be processed by specific metalloproteases to release the soluble bioactive factors from the cell surface. This cleavage is performed by a disintegrin and metalloprotease (ADAM) family members (9, 72, 80, 90) and matrix metalloproteinases (MMPs; Refs. 55, 104) and tightly regulated by different factors. ADAM-dependent EGFR ligand shedding can be induced by activation of G protein-coupled receptors (GPCRs), such as ANG II (ATR) or endothelin-1 (ETR) receptors (Fig. 3). A crucial role for ANG II-dependent EGFR transactivation in chronic kidney disease was demonstrated in mice overexpressing the dominant negative form of EGFR (57). MMPs, a large family of proteolytic enzymes that have the capability to degrade extracellular matrix proteins, are also implicated in regulating nephron formation and the pathogenesis of kidney diseases (13, 107, 121). For instance, it was shown recently that chronic administration of MMP inhibitors delays the progression of, and may even reverse, hypertension and diabetic nephropathy (125).

Fig. 3.

Transactivation of ErbBs by G protein-coupled receptors (GPCR). GPCR activation triggers intracellular signaling cascades that induce transactivation of matrix metalloproteinases (MMPs)/a disintegrin and metalloproteases (ADAMs) family proteins, which are able to cleave ErbB ligands into soluble active moieties. EGF family ligands can then bind to and activate ErbB and facilitate receptor dimerization and downstream activation of the intracellular protein kinases cascades of the same or of the other cell.

Activation of ErbB receptors by binding to their ligands promotes several biological responses. The physiological role of ErbB receptors is well established, especially in cancer. What is more, the role of ErbB-family members in renal development, physiology, and pathophysiology is also well recognized (130). Under physiological conditions, ErbB activation appears to play an important role in the regulation of renal hemodynamics and electrolyte handling by the kidney, while in different pathophysiological states ErbB activation may mediate either beneficial or detrimental effects to the kidney (130). ErbB signaling is critically involved in cell signaling, cell growth, proliferation, and renal electrolyte homeostasis. Once activated by site-specific phosphorylation, ErbB receptors serve as molecular integrators through either direct phosphorylation of target molecules or by serving as scaffolds for adaptor proteins. The great diversity of ligands and receptor dimer pairs allows activation of numerous signaling pathways that coordinately regulate complex processes including developmental growth control and adult homeostasis.

Effects of EGF-Family Members in the Kidney

The involvement of EGFR signaling in AKI and CKD development has been recently thoroughly reviewed (108). Although the precise mechanisms underlying effects of EGF signaling are not completely clear, numerous studies evidence abnormal EGF-EGFR axis functionality during progression and development of these diseases. Changes in EGFR expression and EGFR phosphorylation were detected in a variety of experimental models of AKI (15, 41, 44, 108) and CKD (16, 42, 56, 63, 109). Pivotal role of HB-EGF expression and EGFR signaling is also demonstrated in rapidly progressive glomerulonephritis (10, 31); abnormalities of apico-basal polarity and expression of EGF-EGFR axis were also described in the PKD epithelia (68, 126, 133).

What is more, EGF-family growth factors are involved in regulation of various epithelial ion channels in the kidney. For instance, EGF stimulates store-operated Ca²⁺ channels in human mesangial cells through an intracellular signaling mechanism involving tyrosine kinase and PKC (66). Later it was shown that EGF activates store-operated Ca²⁺ channels by a PLC-dependent, but inositol 1,4,5-trisphosphate receptor-independent, pathway (60). Polycistin-2, critical in PKD, can be also activated in response to EGF in the kidney epithelial cells (67). The physiological relevance of EGF-induced activation of TRPP2 was supported by animal studies in which homozygous deletion of the Egfr gene resulted in cystic dilatation of collecting ducts (112, 113). A role for ErbB receptor activation in the regulation of Mg²⁺ channels in the kidney has also been demonstrated. Genetic analysis has revealed that the melastatin transient receptor potential 6 (TRPM6) channel is mutated in patients with primary hypomagnesemia and secondary hypocalcemia (92, 122). Furthermore, the direct effects of EGF on TRPM6 channels were reported (46, 47, 110) and it was demonstrated that the EGFR inhibitor erlotinib is capable of affecting TRPM6 regulation and thereby altering Mg²⁺ handling in vivo (22).

EGF-Dependent Regulation of Epithelial Na⁺ Channel-Mediated Sodium Transport in the Kidney

The epithelial Na⁺ channel (ENaC) activity is the rate-limiting step for Na⁺ reabsorption across many epithelia, including the distal nephron (7, 101, 102). Dysfunction and aberrant regulation of this channel lead to a spectrum of diseases including hyper- and hypotension. For at least two decades, EGF has been known to regulate ENaC-mediated sodium transport. It was previously shown that EGF decreases active sodium transport in the isolated and perfused rabbit CD (75, 119, 120, 123) and immortalized epithelial cells (25, 26, 37, 94). Interestingly, EGF-family members cause a biphasic response of ENaC. EGF transiently increases sodium transport in Xenopus laevis kidney A6 cells in a phosphatidylinositol 3-kinase-dependent manner, and this stimulation is mediated by the inhibition of the MAPK pathway (70). Later it was reported that acute treatment with EGF and TGF-α enhanced ENaC activity in A6 cells by affecting channel open probability via phosphatidylinositol 3-kinase signaling pathway (62). However, the same study demonstrated that continuous treatment with EGF and TGF-α inhibited ENaC activity by decreasing the number of channels in the plasma membrane through MAPK1/2 pathways (62). Our data are also consistent with the idea that EGF-family members have a biphasic effect on sodium absorption in the mammalian kidney (59). Four members of the EGF-family (EGF, TGF-α, HB-EGF, and amphiregulin) were tested in cultured mpkCCD_c14 principal cells. Basolateral addition of growth factors to these cells initially increased Na⁺ reabsorption above basal levels in a time-dependent manner. A significant increase was detected after 30 min, the earliest time point measured, with a maximum reached by 2 h. After 4 h of treatment, a slow and continuous decrease of ENaC-mediated transepithelial current was observed. Thus chronic treatment of monolayers with EGF-family growth factors leads to significant inhibition of ENaC-mediated current (59).

EGF-family ligands, as many other signaling molecules, activate corresponding receptors in a dose-responded manner. Thus establishing the dose-response curves for the effects of EGF and other members of this family is critical for understanding physiological role of these growth factors. As we previously demonstrated, using immortalized mouse mpkCCD_c14 principal cells, both EGF and TGF-α dose dependently modulated ENaC activity. The EC₅₀ value for EGF activation (acute phase) was 3.7 ± 1.5 ng/ml. Chronic treatment with various concentrations of EGF (0.1, 0.5, 1, 10, and 100 ng/ml) and TGF-α (0.5, 1, 10, and 100 ng/ml) also resulted in dose-dependent decreases in ENaC-mediated transepithelial current (59). Liu et al. (62) also reported that EGF-family ligands have dose-dependent biphasic effects on amphibian A6 cells. Both TGF-α and EGF chronically decreased sodium transport in a dose-dependent manner. The IC₅₀ was 4.7 ng/ml for TGF-α-induced inhibition and 110 ng/ml for EGF-induced inhibition (62). A number of studies analyzed concentrations of EGF-family ligands in plasma and urine of rodents and human. For instance, it was shown that concentrations of EGF in serum of 60-day-old male mice ranged between 0.264 and 0.503 ng/ml (84); significant age and gender variability of EGF level was also noted by the authors. Considering the fact that ErbB receptors localize at the basolateral side and concentration in the lumen is at least 10-fold higher, these concentrations could represent the same range as levels required for ENaC modulation.

At least acute effects of EGF correlate with reactive oxygen species (ROS) production since pretreatment with the nonselective NADPH oxidase activity inhibitor apocynin blunted both generation of ROS and increase in ENaC-mediated current in response to EGF (48). Most likely, an increase in hydrogen peroxide (H₂O₂) levels mediates this effects since it was shown that H₂O₂ is critical for ENaC activity (23, 65, 69, 103). Furthermore, EGF had no effect in Rac1 knockdowned principal cells (48). It appears that small GTPase Rac1, which has been shown to be implicated in the development of many renal and cardiovascular diseases (54, 76, 95, 96), is a critical protein in transmission of the signal from EGF to ENaC. Rac1 might mediate its effects on ENaC either through the MAPK pathway (62, 100), WAVE proteins (52), or ROS production (48), since Rac1 is also one of the key subunits of the NADPH oxidase complex.

Role of EGF-EGFR Axis in the Development of Hypertension

A link between EGF-EGFR signaling pathway and renal vasculature and nephron functions was identified in several studies. Previous studies revealed that renal cortical expression of EGFR was increased in both prehypertensive and hypertensive salt-sensitive (SS) rats (129). Immunohistochemical analysis demonstrated that EGFR expression was increased in SS rats compared with Sprague-Dawley and Dahl/Rapp salt-resistant rats. Following addition of EGF, autophosphorylation of the EGFR was further enhanced in the primary cells derived from SS rats and this effect was especially strengthened in the renal vasculature (129). Previous EGF binding studies also revealed that SS rats (105) and the spontaneously hypertensive Lyon rats exhibited increased levels of EGFR in freshly prepared kidney and aortic tissue membranes compared with normotensive or hypotensive strains (106). Microarray-based global gene expression analysis in deoxycorticosterone acetate (DOCA)-salt-induced hypertensive rats further demonstrated that chronic treatment with an EGFR inhibitor AG1478 leads to inhibition of the majority of genes associated with development of renal dysfunction and hypertension in this model (5). Thus expression of EGFR is associated with hypertensive pathology and can potentially play a role in kidney damage associated with high blood pressure. However, it is hard to define whether changes in gene/protein expression are a consequence of hypertension or the primary basis of the diseases. Previous excellent review by Cowley and Roman (19) discussed potential lines of evidence, which could help to evaluate this question. Additional studies are required for better understanding of the mechanisms resulting in development of hypertension, including salt-sensitive hypertension.

Several possible mechanisms may account for the role of ErbB signaling in the development of hypertension (4). EGF induces constriction of both preglomerular and postglomerular arterioles, resulting in acute major reductions in the rates of glomerular filtration and perfusion (40). EGF and TGF were described as vasoconstrictors on endothelium-denuded thoracic aortas dissected from two models of hypertension: 1) DOCA-salt; and 2) one-kidney, one-clip hypertensive rats (32). HB-EGF was also characterized as a vasoconstrictor released under influence of GPCRs, such as adrenoceptors and angiotensin receptors, in isolated mesenteric arteries (38). Similarly, HB-EGF is required for endothelin-1-induced vasoconstriction and increase in blood pressure (14). Transactivation of EGFRs by GPCRs was also shown in the thoracic aorta smooth muscle cell line (24) and rat aorta (115). Moreover, it was shown that ANG II-induced hypertension and hypertrophy are attenuated by EGFR antisense (50). Briefly, a number of lines of evidence suggest that EGF-EFGR signaling mediates vasoconstriction and arterial hypertension in response to various stimuli. However, most of the studies of the role of EGF-EGFR axis in the vasculature are performed in the heart and conduit vessels and outside of the focus of current review.

As discussed above, in the collecting ducts EGF has a biphasic effect on ENaC-mediated sodium reabsorption. While acute changes to ErbB ligands are critical for the fast regulation of channel activity in response to alterations in cells environment under certain pathological conditions, chronic inhibition of ENaC is relevant to changes in the blood pressure and development of hypertension. Our recent data indicate that ENaC contributes to the development of hypertension in the Dahl SS rat strain (82), which is a commonly used model for the studies of salt-sensitive hypertension (20, 21, 28, 45, 132). Although a high-salt diet was found to downregulate ENaC in salt-resistant animals (33, 86), it was previously demonstrated that ENaC expression is upregulated in SS rats (1–3, 51). Our studies provide evidence that ENaC is abnormally regulated by dietary sodium in SS hypertensive rats, and this abnormal activity is one of the major factors causing salt-sensitive hypertension. Thus, as we recently demonstrated, ENaC activity is significantly enhanced in SS rats fed a high-salt diet compared with salt-resistant SS.13^BN consomic strain and SS rats fed a low-salt diet (82). Importantly, the servo-controlled approach, which allowed us to determine the contribution of renal perfusion pressure (RPP) in the development of hypertension and renal injury in SS rats, provides evidence that ENaC is essential for the development of salt-sensitive hypertension. In the servo-controlled studies, the system maintains the RPP to the left kidney of the instrumented SS rat at control level, whereas RPP to the right kidney increases in response to a high-salt diet. Therefore, both left and right kidneys are exposed to an identical systemic neurohormonal and metabolic environment, but controlled levels of RPP within the left kidney protected it from the high pressure. At least, β-ENaC expression (as analyzed by immunohistochemistry) was increased in the uncontrolled kidneys compared with controlled left kidneys (82).

Our recent studies identified EGF as a key molecular substrate for a new positive feedback mechanism that diminishes development of salt-sensitive hypertension (82). First, we found that EGF concentration in the kidney cortex of SS rats fed a high-salt diet was significantly lower compared with SS rats on a low-salt diet or salt-resistant SS.13^BN consomic rats on both diets (82), which is consistent with upregulation of EGFR expression (105, 129). To directly evaluate the role of EGF in the development of hypertension and its effect on ENaC activity, EGF was intravenously infused and blood pressure was continuously monitored. In addition, ENaC activity was assessed at the end of experiments, when EGF was continuously infused during several days. Infusion of EGF decreased ENaC activity, prevented the development of hypertension and attenuated renal glomerular and tubular damage (82). As shown on Fig. 4, in vivo EGF infusion prevents development of salt-induced hypertension (Fig. 4A) in this strain and decreases ENaC activity in collecting ducts, which might mediate EGF effects on blood pressure (Fig. 4, B and C). We believe that these findings advance our understanding of salt-sensitive hypertension and provide some insight into its molecular basis (Fig. 5). Importantly, recent studies in mice demonstrated that the EGFR monoclonal antibody Cetuximab given intraperitoneally for 8–10 days raised the blood pressure of mice on normal-salt diet by 25 mmHg and that of those on high-salt diets by 34 mmHg (85). The authors proposed that downregulation of ENaC by arachidonic acid metabolites (83, 124) mediates these effects (85). Thus it appears that EGF-EGFR signaling is involved in blood pressure regulation not only in salt-sensitive but also in salt-resistant animals.

Fig. 4.

Chronic infusion of EGF prevents development of hypertension in salt-sensitive (SS) rats fed a high-salt diet via downregulation of ENaC activity. A: effect of continuous intravenous EGF infusion on the mean arterial pressure in the SS rats fed a high-salt diet. B and C: representative single channel traces (B) and summary graph (C) for ENaC activity from freshly isolated split opened collecting ducts derived from SS rats after chronic treatment with EGF or vehicle. MAP, mean arterial pressure; NP_o, average channel activity. [Reproduced from Ref. 82 with permission].

Fig. 5.

Scheme illustrating the hypothesized mechanisms responsible for involvement of EGF and ENaC in development of salt-sensitive hypertension.

Conclusion

ErbB family members are implicated in the development of cancer and cardiac and renal diseases such as AKI, CKD, PKD, and hypertension. Therefore, the therapeutic potential of targeting the ErbB receptors and EGF-family signaling pathways needs further detailed investigations. Undoubtedly, many questions remain regarding regulation of sodium reabsorption and hypertension by ErbB ligands. For instance, receptor specificity plays the key role in these mechanisms. Furthermore, it is not clear if different ligands targeting the same homo- or heteromer work as a complex and whether they have different affinities and different efficacies depending on the target conformation. Also, transactivation of ErbB receptors and the crucial roles of ADAM and MMP proteases are understudied. In addition, we would like to emphasize that special caution should be paid during the treatment of cancer patients with antibodies vs. ErbBs of inhibitors of EGF-ErbB signaling since inhibition of EGF-ErbBs axis could result in development of hypertension as a side effect in patients with predisposition to salt-sensitive hypertension.

GRANTS

Part of this work was supported by National Heart, Lung, and Blood Institute Grant HL-108880 (to A. Staruschenko) and American Heart Association Grant 13SDG14220012 (to T. S. Pavlov).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: A.S. conception and design of research; A.S. drafted manuscript; A.S., O.P., D.V.I., and T.S.P. edited and revised manuscript; A.S., O.P., D.V.I., and T.S.P. approved final version of manuscript; O.P. and D.V.I. prepared figures.