Cellular Metabolism of Substance P Produces Neurokinin-1 Receptor Peptide Agonists with diminished cyclic AMP Signaling.

Substance P (SP) is released from sensory nerves in arteries and heart. It activates neurokinin-1 receptors (NK1R) causing vasodilation, immune modulation, and adverse cardiac remodeling. The hypothesis was tested that SP and SP metabolites activate different second messenger signaling pathways. Macrophages, endothelial cells and fibroblasts metabolized SP to N- and C-terminal metabolites to varying extents. SP 5-11 was the most abundant metabolite followed by SP 1-4, SP 7-11, SP 6-11, SP 3-11 and SP 8-11. In NK1R expressing HEK293 cells, SP and some C-terminal SP metabolites stimulate the NK1R promoting dissociation of several Ga proteins including Gas and Gaq from their bg subunits. SP increases intracellular calcium concentrations ([Ca]i) and cyclic AMP (cAMP) accumulation with similar -log EC50 values of 8.5±0.3 and 7.8±0.1 M, respectively. N-Terminal metabolism of SP by up to 5 amino acids and C-terminal deamidation of SP produce peptides that retain activity to increase [Ca]i but not to increase cAMP. C-Terminal metabolism results in loss of both activities. Thus, [Ca]i and cAMP signaling are differentially affected by SP metabolism. To assess the role of N-terminal metabolism, SP and SP 6-11 were compared on cAMP-mediated activities in NK1R expressing 3T3 fibroblasts. SP inhibits NFkB activity, cell proliferation and wound healing and stimulates collagen production. SP 6-11 had little or no activity. COX-2 expression is increased by SP but not SP 6-11. Thus, metabolism may select the cellular response to SP by inhibiting or re-directing the second messenger signaling pathway activated by the NK1R.

Replacement substance P reduces cardiac fibrosis in monkeys with type 2 diabetes.

BACKGROUND: Type 2 diabetes mellitus (T2DM)-associated cardiac fibrosis contributes to heart failure. We previously showed that diabetic mice with cardiomyopathy, including cardiac fibrosis, exhibit low levels of the neuropeptide substance P; exogenous replacement of substance P reversed cardiac fibrosis, independent of body weight, blood glucose and blood pressure. We sought to elucidate the effectiveness and safety of replacement substance P to ameliorate or reverse cardiac fibrosis in type 2 diabetic monkeys. METHODS: Four female T2DM African Green monkeys receive substance P (0.5 mg/Kg/day S.Q. injection) for 8 weeks. We obtained cardiac magnetic resonance imaging and blood samples to assess left ventricular function and fibrosis by T1 map-derived extracellular volume as well as circulating procollagen type I C-terminal propeptide. Hematological parameters for toxicities were also assessed in these monkeys and compared with three female T2DM monkeys receiving saline S.Q. as a safety comparison group. RESULTS: Diabetic monkeys receiving replacement substance P exhibited a ∼20% decrease in extracellular volume (p = 0.01), concomitant with ∼25% decrease procollagen type I C-terminal propeptide levels (p = 0.008). Left ventricular ejection fraction was unchanged with substance P (p = 0.42); however, circumferential strain was improved (p < 0.01). Complete blood counts, glycosylated hemoglobin A1c, lipids, liver and pancreatic enzymes, and inflammation markers were unchanged (p > 0.05). CONCLUSIONS: Replacement substance P reversed cardiac fibrosis in a large preclinical model of type 2 diabetes, independent of glycemic control. No hematological or organ-related toxicity was associated with replacement substance P. These results strongly support a potential application for replacement substance P as safe therapy for diabetic cardiac fibrosis.

Foxe1 Deletion in the Adult Mouse Is Associated With Increased Thyroidal Mast Cells and Hypothyroidism.

CONTEXT: Foxe1 is a key thyroid developmental transcription factor. Germline deletion results in athyreosis and congenital hypothyroidism. Some data suggest an ongoing role for maintaining thyroid differentiation. OBJECTIVE: We created a mouse model to directly examine the role of Foxe1 in the adult thyroid. METHODS: A model of tamoxifen-inducible Cre-mediated ubiquitous deletion of Foxe1 was generated in mice of C57BL/6J background (Foxe1flox/flox/Cre-TAM). Tamoxifen or vehicle was administered to Foxe1flox/flox/Cre mice aged 6-8 weeks. Blood was collected at 4, 12, and 20 weeks, and tissues after 12 or 20 weeks for molecular and histological analyses. Plasma total thyroxine (T4), triiodothyronine, and thyrotropin (TSH) were measured. Transcriptomics was performed using microarray or RNA-seq and validated by reverse transcription quantitative polymerase chain reaction. RESULTS: Foxe1 was decreased by approximately 80% in Foxe1flox/flox/Cre-TAM mice and confirmed by immunohistochemistry. Foxe1 deletion was associated with abnormal follicular architecture and smaller follicle size at 12 and 20 weeks. Plasma TSH was elevated in Foxe1flox/flox/Cre-TAM mice as early as 4 weeks and T4 was lower in pooled samples from 12 and 20 weeks. Foxe1 deletion was also associated with an increase in thyroidal mast cells. Transcriptomic analyses found decreased Tpo and Tg and upregulated mast cell markers Mcpt4 and Ctsg in Foxe1flox/flox/Cre-TAM mice. CONCLUSION: Foxe1 deletion in adult mice was associated with disruption in thyroid follicular architecture accompanied by biochemical hypothyroidism, confirming its role in maintenance of thyroid differentiation. An unanticipated finding was an increase in thyroidal mast cells. These data suggest a possible explanation for previous human genetic studies associating alleles in/near FOXE1 with hypothyroidism and/or autoimmune thyroiditis.

Relaxin/serelaxin for cardiac dysfunction and heart failure in hypertension.

The pregnancy related hormone relaxin is produced throughout the reproductive system. However, relaxin also has important cardiovascular effects as part of the adaptation that the cardiovascular system undergoes in response to the extra demands of pregnancy. These effects are primarily mediated by the relaxin family peptide receptor 1, which is one of four known relaxin receptors. The effects of relaxin on the cardiovascular system during pregnancy, as well as its anti-fibrotic and anti-inflammatory properties, have led to extensive studies into the potential of relaxin therapy as an approach to treat heart failure. Cardiomyocytes, cardiac fibroblasts, and endothelial cells all possess relaxin family peptide receptor 1, allowing for direct effects of therapeutic relaxin on the heart. Many pre-clinical animal studies have demonstrated a beneficial effect of exogenous relaxin on adverse cardiac remodeling including inflammation, fibrosis, cardiomyocyte hypertrophy and apoptosis, as well as effects on cardiac contractile function. Despite this, clinical studies have yielded disappointing results for the synthetic seralaxin, even though seralaxin was well tolerated. This article will provide background on relaxin in the context of normal physiology, as well as the role of relaxin in pregnancy-related adaptations of the cardiovascular system. We will also present evidence from pre-clinical animal studies that demonstrate the potential benefits of relaxin therapy, as well as discussing the results from clinical trials. Finally, we will discuss possible reasons for the failure of these clinical trials as well as steps being taken to potentially improve relaxin therapy for heart failure.

Histamine receptors in heart failure.

The biogenic amine, histamine, is found predominantly in mast cells, as well as specific histaminergic neurons. Histamine exerts its many and varied actions via four G-protein-coupled receptors numbered one through four. Histamine has multiple effects on cardiac physiology, mainly via the histamine 1 and 2 receptors, which on a simplified level have opposing effects on heart rate, force of contraction, and coronary vasculature function. In heart failure, the actions of the histamine receptors are complex, the histamine 1 receptor appears to have detrimental actions predominantly in the coronary vasculature, while the histamine 2 receptor mediates adverse effects on cardiac remodeling via actions on cardiomyocytes, fibroblasts, and even endothelial cells. Conversely, there is growing evidence that the histamine 3 receptor exerts protective actions when activated. Little is known about the histamine 4 receptor in heart failure. Targeting histamine receptors as a therapeutic approach for heart failure is an important area of investigation given the over-the-counter access to many compounds targeting these receptors, and thus the relatively straight forward possibility of drug repurposing. In this review, we briefly describe histamine receptor signaling and the actions of each histamine receptor in normal cardiac physiology, before describing in more detail the known role of each histamine receptor in adverse cardiac remodeling and heart failure. This includes information from both clinical studies and experimental animal models. It is the goal of this review article to bring more focus to the possibility of targeting histamine receptors as therapy for heart failure.

Replacement of Lost Substance P Reduces Fibrosis in the Diabetic Heart by Preventing Adverse Fibroblast and Macrophage Phenotype Changes.

Reduced levels of the sensory nerve neuropeptide substance P (SP) have been reported in the diabetic rat heart, the consequence being a loss of cardioprotection in response to ischemic post-conditioning. We considered whether this loss of SP also predisposes the heart to non-ischemic diabetic cardiomyopathy in the form of fibrosis and hypertrophy. We report that diabetic Leprdb/db mice have reduced serum SP and that administration of exogenous replacement SP ameliorated cardiac fibrosis. Cardiac hypertrophy did not occur in Leprdb/db mice. Cardiac fibroblasts exposed to high glucose converted to a myofibroblast phenotype and produced excess extracellular matrix proteins; this was prevented by the presence of SP in the culture media. Cardiac fibroblasts exposed to high glucose produced increased amounts of the receptor for advanced glycation end products, reactive oxygen species and inflammatory cytokines, all of which were prevented by SP. Cultured macrophages assumed an M1 pro-inflammatory phenotype in response to high glucose as indicated by increased TNF-α, CCL2, and IL-6. SP promoted a shift to the reparative M2 macrophage phenotype characterized by arginase-1 and IL-10. Leprdb/db mice showed increased left ventricular M1 phenotype macrophages and an increase in the M1/M2 ratio. Replacement SP in Leprdb/db mice restored a favorable M1 to M2 balance. Together these findings indicate that a loss of SP predisposes the diabetic heart to developing fibrosis. The anti-fibrotic actions of replacement SP involve direct effects on cardiac fibroblasts and macrophages to oppose adverse phenotype changes. This study identifies the potential of replacement SP to treat diabetic cardiomyopathy.

The Histamine 3 Receptor Is Expressed in the Heart and Its Activation Opposes Adverse Cardiac Remodeling in the Angiotensin II Mouse Model.

Histamine is a basic amine stored in mast cells, with its release capable of activating one of four histamine receptors. The histamine 3 receptor (H3R) is known to be cardioprotective during acute ischemia by acting to limit norepinephrine release. However, a recent study reported that myofibroblasts isolated from the infarct zone of rat hearts responded to H3R activation by up-regulating collagen production. Thus, it is necessary to clarify the potential role of the H3R in relation to fibrosis in the heart. We identified that the mouse left ventricle (LV) expresses the H3R. Isolation of mouse cardiac fibroblasts determined that while angiotensin II (Ang II) increased levels of the H3R, these cells did not produce excess collagen in response to H3R activation. Using the Ang II mouse model of adverse cardiac remodeling, we found that while H3R blockade had little effect on cardiac fibrosis, activation of the H3R reduced cardiac fibrosis and macrophage infiltration. These findings suggest that when activated, the H3R is anti-inflammatory and anti-fibrotic in the mouse heart and may be a promising target for protecting against cardiac fibrosis.

The Diabetic Cardiac Fibroblast: Mechanisms Underlying Phenotype and Function.

Diabetic cardiomyopathy involves remodeling of the heart in response to diabetes that includes microvascular damage, cardiomyocyte hypertrophy, and cardiac fibrosis. Cardiac fibrosis is a major contributor to diastolic dysfunction that can ultimately result in heart failure with preserved ejection fraction. Cardiac fibroblasts are the final effector cell in the process of cardiac fibrosis. This review article aims to describe the cardiac fibroblast phenotype in response to high-glucose conditions that mimic the diabetic state, as well as to explain the pathways underlying this phenotype. As such, this review focuses on studies conducted on isolated cardiac fibroblasts. We also describe molecules that appear to oppose the pro-fibrotic actions of high glucose on cardiac fibroblasts. This represents a major gap in knowledge in the field that needs to be addressed.

Regulation of Cardiac Mast Cell Maturation and Function by the Neurokinin-1 Receptor in the Fibrotic Heart.

Cardiac fibrosis is an underlying cause of diastolic dysfunction, contributing to heart failure. Substance P (SP) activation of the neurokinin-1 receptor (NK-1R) contributes to cardiac fibrosis in hypertension. However, based on in vitro experiments, this does not appear to be via direct activation of cardiac fibroblasts. While numerous cells could mediate the fibrotic effects of SP, herein, we investigate mast cells (MC) as a mechanism mediating the fibrotic actions of SP, since MCs are known to play a role in cardiac fibrosis and respond to SP. Spontaneously hypertensive rats (SHR) were treated with the NK-1R antagonist L732138 (5 mg/kg/d) from 8 to 12 weeks of age. L732138 prevented increased MC maturation of resident immature MCs. NK-1R blockade also prevented increased cardiac MC maturation in angiotensin II-infused mice. MC-deficient mice were used to test the importance of MC NK-1Rs to MC activation. MC-deficient mice administered angiotensin II did not develop fibrosis; MC-deficient mice reconstituted with MCs did develop fibrosis. MC-deficient mice reconstituted with MCs lacking the NK-1R also developed fibrosis, indicating that NK-1Rs are not required for MC activation in this setting. In conclusion, the NK-1R causes MC maturation, however, other stimuli are required to activate MCs to cause fibrosis.

Substance P-mediated cardiac mast cell activation: An in vitro study.

The neuropeptide substance P can induce degranulation of cardiac mast cells at high concentrations. Herein, we seek to further understand substance P activation of cardiac mast cells in the context of other neuropeptides as well as modulation by non-neuropeptides. This is important given the increasingly recognized role of both cardiac mast cells and substance P in adverse cardiac remodeling. To address this, we isolated cardiac mast cells and compared their response to substance P as well as other members from the tachykinin family of peptides, including neurokinin A and hemokinin-1. We also tested the ability of other factors to manipulate the cardiac mast cell response to substance P. We found that while neurokinin A did not induce cardiac mast cell degranulation, both substance P and hemokinin-1 induced a concentration-dependent release of histamine; the maximal response to hemokinin-1 was greater than to substance P. Neurokinin-1 receptor blockade prevented substance P-induced histamine release, while only partially attenuating hemokinin-1-induced histamine release. The antioxidant N-acetylcysteine attenuated histamine release in response to hemokinin-1 and had no effect on substance P-induced histamine release. Selective PPAR-γ agonists attenuated histamine release in response to substance P. These data indicate that substance P activates cardiac mast cells via the neurokinin-1 receptor, and that the activation response is different to other tachykinins. That the response to substance P is receptor mediated and can be modulated by activation of other receptors (PPAR-γ), argues that substance P activation of cardiac mast cells has potential biological significance.

Doxorubicin-Induced Myocardial Fibrosis Involves the Neurokinin-1 Receptor and Direct Effects on Cardiac Fibroblasts.

BACKGROUND: Cancer patients receiving anthracycline-based chemotherapy (Anth-bC) may experience early cardiac fibrosis, which could be an important contributing mechanism to the development of impaired left ventricular (LV) function. Substance P, a neuropeptide that predominantly acts via the neurokinin 1 receptor (NK-1R), contributes to adverse myocardial remodelling and fibrosis in other cardiomyopathies. We sought to determine if NK-1R blockade is effective against doxorubicin (Dox - a frequently used Anth-bC)-induced cardiac fibrosis and cardiomyocyte apoptosis. In addition, we explored the direct effects of Dox on cardiac fibroblasts. METHODS: Male Sprague-Dawley rats were randomised to receive saline, six cycles of Dox (1.5mg Dox/kg/cycle) or Dox with an NK-1R antagonist (L732138, 5mg/kg/daily through Dox treatment). At 8 weeks after the initial dose of Dox, LV function and histopathological myocardial fibrosis and cell apoptosis were assessed. Collagen secretion was measured in vitro to test direct Dox activation of cardiac fibroblasts. RESULTS: Rats undergoing Dox treatment (9mg/kg cumulative dose) developed cardiac fibrosis and cardiomyocyte apoptosis. NK-1R blockade partially mitigated cardiac fibrosis while completely preventing cardiomyocyte apoptosis. This resulted in improved diastolic function. Furthermore, we found that Dox had direct effects on cardiac fibroblasts to cause increased collagen production and enhanced cell survival. CONCLUSIONS: This study demonstrates that cardiac fibrosis induced by Anth-bC can be reduced by NK-1R blockade. The residual fibrotic response is likely due to direct Dox effects on cardiac fibroblasts to produce collagen.

Mast Cells: Key Contributors to Cardiac Fibrosis.

Historically, increased numbers of mast cells have been associated with fibrosis in numerous cardiac pathologies, implicating mast cells in the development of cardiac fibrosis. Subsequently, several approaches have been utilised to demonstrate a causal role for mast cells in animal models of cardiac fibrosis including mast cell stabilising compounds, rodents deficient in mast cells, and inhibition of the actions of mast cell-specific proteases such as chymase and tryptase. Whilst most evidence supports a pro-fibrotic role for mast cells, there is evidence that in some settings these cells can oppose fibrosis. A major gap in our current understanding of cardiac mast cell function is identification of the stimuli that activate these cells causing them to promote a pro-fibrotic environment. This review will present the evidence linking mast cells to cardiac fibrosis, as well as discuss the major questions that remain in understanding how mast cells contribute to cardiac fibrosis.

The role of neuropeptides in adverse myocardial remodeling and heart failure.

In addition to traditional neurotransmitters of the sympathetic and parasympathetic nervous systems, the heart also contains numerous neuropeptides. These neuropeptides not only modulate the effects of neurotransmitters, but also have independent effects on cardiac function. While in most cases the physiological actions of these neuropeptides are well defined, their contributions to cardiac pathology are less appreciated. Some neuropeptides are cardioprotective, some promote adverse cardiac remodeling and heart failure, and in the case of others their functions are unclear. Some have both cardioprotective and adverse effects depending on the specific cardiac pathology and progression of that pathology. In this review, we briefly describe the actions of several neuropeptides on normal cardiac physiology, before describing in more detail their role in adverse cardiac remodeling and heart failure. It is our goal to bring more focus toward understanding the contribution of neuropeptides to the pathogenesis of heart failure, and to consider them as potential therapeutic targets.

Novel Omega-3 Fatty Acid Epoxygenase Metabolite Reduces Kidney Fibrosis.

Cytochrome P450 (CYP) monooxygenases epoxidize the omega-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid into novel epoxydocosapentaenoic acids (EDPs) that have multiple biological actions. The present study determined the ability of the most abundant EDP regioisomer, 19,20-EDP to reduce kidney injury in an experimental unilateral ureteral obstruction (UUO) renal fibrosis mouse model. Mice with UUO developed kidney tubular injury and interstitial fibrosis. UUO mice had elevated kidney hydroxyproline content and five-times greater collagen positive fibrotic area than sham control mice. 19,20-EDP treatment to UUO mice for 10 days reduced renal fibrosis with a 40%-50% reduction in collagen positive area and hydroxyproline content. There was a six-fold increase in kidney α-smooth muscle actin (α-SMA) positive area in UUO mice compared to sham control mice, and 19,20-EDP treatment to UUO mice decreased α-SMA immunopositive area by 60%. UUO mice demonstrated renal epithelial-to-mesenchymal transition (EMT) with reduced expression of the epithelial marker E-cadherin and elevated expression of multiple mesenchymal markers (FSP-1, α-SMA, and desmin). Interestingly, 19,20-EDP treatment reduced renal EMT in UUO by decreasing mesenchymal and increasing epithelial marker expression. Overall, we demonstrate that a novel omega-3 fatty acid metabolite 19,20-EDP, prevents UUO-induced renal fibrosis in mice by reducing renal EMT.

THE AUTOCRINE ROLE OF TRYPTASE IN PRESSURE OVERLOAD-INDUCED MAST CELL ACTIVATION, CHYMASE RELEASE AND CARDIAC FIBROSIS.

BACKGROUND: Cardiac mast cell (MC) proteases, chymase and tryptase, increase proliferation and collagen synthesis in cultured cardiac fibroblasts. However, the question as to why preventing individually the actions of either protease prevents fibrosis when both are released upon MC activation remains unanswered. Since tryptase has the ability to activate MCs in noncardiac tissues via the protease-activated receptor-2 (PAR-2), there is the possibility that its, in vivo, fibrotic role is due to its ability to induce MC degranulation thereby amplifying the release of chymase. METHODS: This study sought to delineate the interactions between tryptase and chymase in myocardial remodeling secondary to transverse aortic constriction (TAC) for 5 wks in male Sprague Dawley rats untreated or treated with either the tryptase inhibitor, nafamostat mesilate or MC membrane stabilizing drug, nedocromil (n=6/group). In addition, ventricular slices from 6 rat hearts were incubated with tryptase, tryptase plus nafamostat mesilate or chymostatin for 24 h. RESULTS AND CONCLUSION: The results indicate the presence of PAR-2 on MCs and that tryptase inhibition and nedocromil prevented TAC-induced fibrosis and increases in MC density, activation, and chymase release. Tryptase also significantly increased chymase concentration in ventricular tissue culture media, which was prevented by the tryptase inhibitor. Hydroxyproline concentration in culture media was significantly increased with tryptase incubation as compared to the control group and the tryptase group incubated with nafamostat mesilate or chymostatin. We conclude that tryptase contributes to TAC-induced cardiac fibrosis primarily via activation of MCs and the amplified release of chymase.

Substance P induces cardioprotection in ischemia-reperfusion via activation of AKT.

Accumulating evidence indicates that substance P is cardioprotective following ischemia-reperfusion primarily due to its potent coronary vasodilator actions. However, an anti-apoptotic effect of substance P has been observed in tenocytes following ischemia, which involved activation of the AKT pathway. This suggests the possibility that substance P also provides cardioprotection via direct actions to activate AKT in myocardial cells. The purpose of this study was to test the hypothesis that substance P attenuates ischemia-related cell death via direct effects on myocardial cells by activating cell survival pathways. Seven-week-old male Sprague-Dawley rats, anesthetized with intraperitoneal pentobarbital sodium (100 mg/kg), were used. The ability of substance P to prevent cellular damage was assessed following ischemia-reperfusion in an isolated heart preparation and in short-term hypoxia without reperfusion using a left ventricular tissue slice culture preparation. In addition, the NK-1 receptor and AKT involvement was assessed using the NK-1 receptor antagonist L732138 and the AKT inhibitor LY294002. The results indicate that substance P reduced the ischemia-related release of lactate dehydrogenase in both preparations and the degree of apoptosis and necrosis in the hypoxic left ventricular slices, indicating its ability to attenuate cell damage; and induced AKT phosphorylation, with both the AKT inhibitor and NK-1 receptor antagonist preventing the increased phosphorylation of AKT and the ability of substance P to attenuate hypoxic cellular damage. It is concluded that substance P reduces ischemia/hypoxia-induced myocardial cell death by acting directly on cardiac cells to initiate cell survival pathways via the NK-1 receptor and AKT.

Differential Effects of Prevention and Reversal Treatment with Lisinopril on Left Ventricular Remodelling in a Rat Model of Heart Failure.

BACKGROUND: Angiotensin converting enzyme (ACE) inhibitors such as lisinopril, represent the front line pharmacological treatment for heart failure, which is characterised by marked left ventricular (LV) dilatation and hypertrophy. This study sought to determine whether initiating treatment with ACE inhibitors at different stages in the remodelling process would alter the efficacy of treatment. METHODS: To this end, LV size and function were determined in the aortocaval (AV) fistula model of volume overload-induced heart failure. Sprague-Dawley rats were assigned to sham, untreated AV fistula (21 weeks), AV fistula treated with lisinopril (21 weeks), or AV fistula treated with lisinopril from six to 21 weeks post-fistula groups. RESULTS: Administration of lisinopril for the entire 21-week period prevented LV dilatation, attenuated myocardial hypertrophy and prevented changes in myocardial compliance and contractility, whereas delaying initiation of treatment until six weeks post-fistula attenuated LV dilatation and hypertrophy, however, the delayed onset of treatment had no beneficial effect on ventricular compliance or systolic function. CONCLUSIONS: The results demonstrate differential effects that can occur with ACE inhibitors depending on the stage during the remodelling process at which treatment is administered.