Relaxin reverses inflammatory and immune signals in aged hearts
Relaxin reverses inflammatory and immune signals in aged hearts
Brian Martin 1 2
Beth Ann Gabris-Weber 2
Rajiv Reddy 2
Guillermo Romero 0 2
Ansuman Chattopadhyay 2
Guy Salama 1 2
0 Department of Pharmacology & Chemical Biology, University of Pittsburgh , Pittsburgh, PA , United States of America, 4 Molecular Biology Information Service, Health Sciences Library System, University of Pittsburgh , Pittsburgh, PA , United States of America
1 Department of Bioengineering, University of Pittsburgh , Pittsburgh, PA , United States of America, 2 School of Medicine, Heart and Vascular Institute, University of Pittsburgh , Pittsburgh, PA , United States of America
2 Editor: Maria Cristina Vinci, Centro Cardiologico Monzino , ITALY
`Healthy' aging drives structural and functional changes in the heart including maladaptive electrical remodeling, fibrosis and inflammation, which lower the threshold for cardiovascular diseases such as heart failure (HF) and atrial fibrillation (AF). Despite mixed results in recent clinical trials, Relaxin-therapy for 2-days could reduce mortality by 37% at 180-days post-treatment, in patients with acute decompensated HF. Relaxin's short life-span (hours) but long-lasting protective actions led us to test the hypothesis that relaxin acts at a genomic level to reverse maladaptive remodeling in aging and HF. Young (9-month) and aged (24-month), male and female F-344/Brown Norway rats were treated with relaxin (0.4 mg/kg/day) for 2-weeks delivered by subcutaneous osmotic minipumps or with sodium acetate (controls). The genomic effects of aging and relaxin were evaluated by extracting RNA from the left ventricles and analyzing genomic changes by RNAsequencing, Ingenuity Pathway Analysis, MetaCore and tissue immunohistochemistry. We found that aging promotes a native inflammatory response with distinct sex-differences and relaxin suppresses transcription of multiple genes and signaling pathways associated with inflammation and HF in both genders. In addition, aging significantly increased: macrophage infiltration and atrial natriuretic peptide levels in female ventricles, and activation of the complement cascade, whereas relaxin reversed these age-related effects.
Data Availability Statement: The data underlying
this study have been uploaded to NCBI's Gene
Expression Omnibus (GEO), and are accessible via
GEO series number GSE106377 at the following
Funding: This work was supported by Samuel and
Emma Winters Foundation (GS), the University of
Pittsburgh School of Medicine Bridge Funds (GS),
and the Innovator Award from the Vascular
Medicine Institute/Heart and Vascular Institute of
the University of Pittsburgh (GS).
Methods and results
These data support the hypothesis that relaxin alters gene transcription and suppresses
inflammatory pathways and genes associated with HF and aging. Relaxin's suppression of
inflammation and fibrosis supports its potential as a therapy for cardiovascular and
inflammation-related diseases, such as HF, AF and diabetes.
Aging is a major risk factor for atrial fibrillation (AF) [
] and heart failure (HF), [
] which may
reach pandemic proportions due to an increasingly aging population, estimated to reach 70
million people over the age of 65 in the United States by 2030. HF is defined as the inability
of the heart muscle to pump enough blood to meet the energy demands of the body , and
aging is associated with multiple maladaptive vascular and cardiac structural, electrical and
functional changes that increase the susceptibly to HF, AF and other cardiovascular diseases
(CDs). [3, 5] Structural remodeling in aging includes: increases in vascular stiffness, left
ventricular (LV) and aortic wall thickness , fibrosis  and myocyte hypertrophy that promote
diastolic dysfunction, coronary heart disease,  AF  and HF.  In sum, age-related
maladaptive responses lower the threshold for CD development.  Recent evidence posits that
aging brings-on the gradual development of a chronic, low-grade inflammation, termed
ªinflammagingº which is possibly the greatest risk factor for many age-related diseases. 
Chronic inflammation can result from failed resolution of an injury by the body or stimuli
resulting in asymptomatic responses. Inflammation has been linked to multiple CDs
including myocardial infarction, resulting in increased leukocyte infiltration that leads to
pathological left ventricular (LV) remodeling.[
] In addition, complement cascade activity can be
activated in the presence of pathogens or tissue injury and has been linked to higher
hospitalizations and mortality in ischemia/reperfusion studies, [9, 11] and to inflammation associated
with insulin resistance and type II diabetes.  Interleukin (IL)-1 and IL-6 have been linked
to cardiac hypertrophy, reduced contractility, increased arrhythmia susceptibility, increased
C-reactive protein, coronary artery disease  and raise stroke risk, while tumor necrosis
factor alpha (TNFα) is associated with LV dilatation and dysfunction and HF progression. [14±
16] In addition to inflammatory markers of disease, multiple markers are used clinically to
determine HF progression and prognosis. Brain natriuretic peptide (BNP, gene: NPPB) is a
marker of HF, is indicative of hemodynamic wall stress and is a risk factor for AF and HF. [15,
17, 18] Like BNP, atrial natriuretic peptide (ANP, gene: NPPA) is a marker of congestive HF
 and growth differentiation factor (GDF)-15 is implicated in HF and inflammatory and
oxidative stress. [18, 20] Clinical trials that suppressed the inflammatory-related cytokine,
TNFα, have yielded disappointing results in chronic HF patients. TNFα chelation had no
effect on primary endpoints of hospitalization and death, leading investigators to hypothesize
a large degree of redundancy in the immune response related to HF, so that targeting a single
cytokine may prove to be ineffective.  Studies in patients with rheumatoid arthritis using
tocilizumab to block IL-6 signaling through the IL-6 receptor have shown beneficial effects,
though resulted in side-effects including pro-atherogenic dyslipidemia and increased low
density lipoprotein (LDL),  which is associated with increased risk of coronary artery disease.
Although age is a major risk factor for inflammation, AF and HF, [
8, 22, 23
] with few
exceptions [23±25] there is a noticeable paucity of genetic studies on how global gene expression is
altered in normal, `healthy' cardiac aging. Many were done in mice despite the challenge of
comparing age-related gene expression patterns in mice and man. Yet, differences in mice
may be due to a non-uniform rate of aging in mice. since other studies identified aging
gene expression signatures that are conserved in mice, rats and humans, namely, inflammation
and the immune response. [
]. This suggests that genomic studies of inflammation in rodents
may be relevant to the development of therapies for disorders related to age and inflammation.
Furthermore, the challenges of using mouse models of aging are largely dispelled through the
use of the F-344 Brown/Norway rat model from the National Institute of Aging, which has
been developed and used extensively to study the effects of aging due to its homogeneous and
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linear rate of aging, increased cardiac collagen production and increased susceptibility to atrial
fibrillation and systolic and diastolic dysfunction; all hallmarks of human aging. [
7, 26, 27
Relaxin (RLX), a hormone of pregnancy, has shown significant cardiovascular benefits
including a significant reduction in AF susceptibility in aged  and hypertensive [
primarily through a reversal of fibrosis and an increase in voltage gated sodium channels,
Nav1.5, and its current, INa. In phase II clinical trials, RLX reduced 180-day cardiovascular and
all-cause mortality in acute decompensated HF patients after 48-hour RLX infusion; [
however, recent Phase III clinical trials failed to meet the primary endpoint of reduced
mortality in HF patients (https://www.novartis.com/ news release, March 2017), though details are
yet to be published. In spite of these results, RLX has been shown to be a potent anti-fibrotic
through regulation of fibroblast activation, collagen secretion and reductions in pro-fibrotic
transcripts,[5, 31] a suppressor of AF through a combination of structural and electrical
] and to increase vascular compliance and reduce systemic vascular
] In addition, we report here RLX's anti-inflammatory actions, suggesting
that RLX acts on multiple signaling pathways and may be a therapy for various diseases
associated with these pathological conditions. Despite growing evidence of RLX's beneficial effects,
[5, 28, 33±35] the mechanisms underlying these effects remain unclear. In RELAX-AHF trials,
RLX serum concentrations rose rapidly, levelled off after 4 hours of infusion, and decreased
quickly post-infusion. The short lifespan of RLX (~2hrs) suggests that the long-term
benefits of RLX stem from long-lasting genomic effects rather than an improvement of
hemodynamics at the time of infusion.[
] Here, we test the hypothesis that RLX acts at the genomic
level to modify HF and AF related genes and signaling pathways that are activated or inhibited
in `healthy/normal' aging. We show that aging significantly increases gene transcription of
multiple pro-inflammatory and HF related genes and signaling pathways. Significantly, we
show that RLX reverses the effects of aging in both genders through genetic regulation of these
pathways and that aging increases macrophage accumulation in females, but not male left
ventricles, which is reversed by RLX treatment. These data provide evidence in support of RLX as
a therapy for a plethora of inflammatory diseases through its regulation of multiple cytokines
and signaling pathways.
Materials and methods
Young (9-month-old) and aged (24-month-old) male and female F-344/Brown Norway rats
were obtained from the National Institute of Aging. Rats were treated with vehicle (sodium
acetate) or RLX (400 μg/kg/day) for 14 days via subcutaneous osmotic mini pumps. Left
ventricular tissue was snap frozen in liquid nitrogen and stored at -80 degrees Celsius until use.
Studies were performed in accordance with the Guide for the Care and Use of Laboratory
Animals and were approved by the Institutional Animal Care and Use Committee at the
University of Pittsburgh.
Three animals were analyzed from male groups and four animals from female groups
(24-month-old ± RLX, 9-month-old ± RLX). A total of 1 μg of RNA was sent to the University
of Pittsburgh Genomics Core for sequencing and library preparation. Library preparation
was performed using the TruSEQ Stranded Total RNA Sample Preparation Kit (Illumina,
San Diego, CA) according to manufacturer's instructions. Following removal of ribosomal
sequences, RNA was fragmented for 8 minutes and reverse transcription performed. Double
stranded cDNA was subjected to 3' adenylation and ligation of sequencing adapters.
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Sequencing was carried out on a NextSeq 500 (Illumina) for 2 x 75 bp paired end reads.
Loading concentration was 1.4 pM for females and 1.2 pM for males.
Raw transcript data was imported into Biomedical Genomics Workbench 3.0.1 and reads were
mapped to the rat reference genome. Differentially expressed genes (DEGs) used in pathway
analysis were determined between old and young rats with and without RLX using filters to
select genes with Reads Per Kilobase of transcript per Million, RPKM 1, absolute fold change
(FC) > 1.5 using total gene count and a false discovery rate (FDR) 0.05. Fisher's exact test
was used to test for pathway significance. Activation (z-score > 2, orange bars) or inhibition
(z-score < 2, blue bars) of each pathway was determined by Ingenuity Pathway Analysis (IPA)
and is a measure of experimentally determined gene expression changes reported in the
literature. For reporting of individual genes, data are presented as FC with p-value and FDR. DEGs
filtered for pathway analysis were imported into Ingenuity Pathway Analysis and MetaCore to
determine signaling pathways and upstream regulators affected by aging or RLX treatment.
Aging studies were carried out by comparing RNA-seq data from 24-month old untreated
(UNT) vs. 9-month old UNT rats. RLXs effect was measured by comparing 24-month-old
+ RLX vs. 24-month-old UNT rats. The data from this publication have been deposited in
NCBI's Gene Expression Omnibus (GEO), and are accessible via GEO series number
GSE106377 at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE106377.
Male (n = 3-4/group) and Female (n = 5/group) left ventricular tissue were thawed and fixed
in 2% PFA at room temperature and left in 30% sucrose for 24 hours at 4 degrees Celsius. LV
tissue sections (7 μm) were placed in 0.1% Triton 100-x for 10 minutes. Sections were blocked
with 2% BSA for 30 minutes followed by a 90-minute incubation with primary antibodies,
rabbit-anti-F4/80 polyclonal (Thermo Fischer, PA5-21399), mouse-anti-CD4 monoclonal
(BioRad, MCA55GA), and mouse-anti ANP monoclonal (Santa Cruz Biotechnology,
sc515701) antibodies. Sections were washed 3x 5minutes and incubated with secondary
antibodies for 45 min and another wash. Finally, sections were stained for 1 minute with DAPI and
coverslips mounted with gelvatol. Images were taken on an Olympus Fluoview inverted
confocal microscope at 60x magnification. Image parameters were obtained for one image and kept
constant throughout imaging. Images were analyzed using ImageJ software and only circular
staining with a clear association with a nucleus were included in the macrophage and T-cell
analysis. ANOVA was used to determine statistically significant differences between groups
with Tukey's post hoc analysis. Data is presented as mean ± SEM. P<0.05 was considered
Effects of aging and relaxin on biomarkers of AF and HF
In the clinical setting, several markers have been associated with the severity and/or mortality
of AF and HF patients. In a proteomics study of AF, Lind et al  reported an association of
multiple proteins with AF using two cohorts. The primary cohort included both genders and a
validation cohort included only males. It is interesting to note that they reported thirteen AF
associated proteins in the mixed gender cohort, but merely 5 in the male only cohort,
suggesting marked gender differences. Table 1 lists age and RLX-dependent transcription changes of
multiple markers thought to be involved in AF and HF development. [14±18, 38±41]
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RNA-sequencing (RNA-seq) analysis showed that aging and aging plus RLX-treatment
altered with high significance (FDR<0.05) more than 7 genes associated with HF and AF in
the clinical setting. In Table 1, these genes are highlighted in bold and dark shading and genes
exhibiting highly significant changes have double-check marks. In females, aging or RLX
altered atrial natriuretic peptide (NPPA), interferon gamma (INFG) and the LDL receptor
(LDLR). In males, aging or RLX altered IL-1β, IL-6, NPPB, and GDF-15. In addition, several
genes exhibited > 1.5 absolute fold changes with p<0.05, but do not reach the more stringent
criterion of FDR<0.05 and are labeled with a single check mark. RLX significantly reversed
multiple transcripts associated with HF and AF and therefore appears to not only stop the
progression but to reverses the pathological expression of many cytokines.
Atrial natriuretic peptide (ANP), the most differentially expressed gene in female rats, was
selected for further protein expression analysis to confirm that the effects of RLX on gene
expression had functional consequences. In female rats, 9-month-old controls (Fig 1A)
exhibited significantly less ANP than in aged ventricles (Fig 1B). RLX treatment (Fig 1C)
significantly reversed aging's effect on ANP expression. In male rats, there was no significant change
FDR: False Discovery Rate. Multiple test correction technique used in RNA-sequencing data analysis. Underscores indicated gene isoform. Bold: at least one group
shows FDR < 0.05. ns: not significant. Yellow,✓✓: FDR<0.05. Blue,✓: Fold Change > 1.5, p<0.05.
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Fig 1. Protein expression of ANP in female and male heart. A-D. Female. Female aging (B) resulted in upregulation of ANP expression compared to
young controls (A) and was reversed by RLX (C). D. Quantification of ANP expression in female LV. E-H. Male. In male rats, aging (F) did not increase
ANP expression compared to young controls (E), though there was a reduction in ANP with RLX treatment (G) though this did not reach significance
compared to young or aged males. H. Quantification of ANP expression in male LV. Red: ANP. : p<0.0001. Scale bar: 50μm.
in ANP expression in the aged versus the young controls (Fig 1E, 1F and 1H), though there
was a reduction in ANP with RLX treatment (Fig 1G and 1H), that did not reach statistical
significance. These data match closely the gene expression data in Table 1 (NPPA) for ANP
Upstream analysis (Table 2) searches for differentially expressed genes (DEGs) to predict
activation or inhibition of transcription regulators and to determine which molecules can
explain gene expression alterations observed in the data sets. Observed DEGs are compared to
a database of known interactions with transcriptional regulators kept by Ingenuity, with
transcripts grouped together under common regulators. The quantity of genes controlled by a
Upstream analysis of regulators
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FDR: False Discovery Rate. Multiple test correction technique used in RNA-sequencing data analysis. Bold: at least one group shows FDR < 0.05. NS: not significant.
Yellow,✓✓: FDR<0.05. Blue,✓: p<0.05.
given transcriptional regulator and the agreement of direction (+/-) of fold change of each
DEG to that reported in the literature, leads to a prediction of activation or inhibition of those
Consistent with the effects of aging shown in Table 1, NFkB signaling, a crucial pathway
regulating many inflammatory genes, is upregulated in female aging and inhibited by RLX
in both female and male rats. In female rats, IFNγ, IL-6, lipopolysaccharide (LPS) and
transforming growth factor (TGF) β1 are activated in aging and inhibited by RLX. Interestingly,
SMAD7, an inhibitor of TGFβ1 and fibrosis, is inhibited with aging and activated by RLX;
consistent with the anti-fibrosis properties of RLX. In aged male rats, upstream analysis failed to
detect an activation of the genes that were activated in aged female ventricles, yet RLX still
inhibited many of them in aged male ventricles. Note that RLX inhibited several upstream
regulators: hydrogen peroxide, NFkB, TLR4, IFNG, IL-6 and LPS in males and females (Table 2).
Besides classic inflammatory gene expression that change in HF, and aging ± RLX, RLX
altered chemo-attractant genes involved in recruiting inflammatory and immune cells to sites
of injury (Table 3).
Signaling pathway analysis
To identify signaling pathways that are affected by aging and RLX treatment, differentially
expressed genes (DEGs) were filtered as transcripts whose absolute expression was altered by
at least 1.5-fold, had expression values with RPKM (reads per kilobase of transcript per
million) 1, and had a false discovery rate (FDR) < 0.05 (Fig 2).
Application of Ingenuity Pathway Analysis (IPA) software to DEGs showed that female rats
exhibit significant activation of inflammatory and immune signaling pathways with aging (Fig
3A) which are reversed by RLX treatment (Fig 3B). These pathways include: 1)
Calcium7 / 17
Fig 2. Volcano plots of differentially expressed genes in aging ± RLX treatment. A. Female aging resulted in an
overall upregulation of transcripts, with fewer transcripts being downregulated. B. RLX treatment in females caused a
marked suppression of most transcripts with few being upregulated. C. Transcript expression changes in males were
muted compared to females during aging, with an even number of transcripts being up and downregulated. D. RLX
treatment in males reflects that seen in females, with a substantial proportion of transcript alterations being suppressed
compared to upregulated. Blue dots represent individual transcripts.
induced T lymphocyte apoptosis pathway, which acts to counter excessive inflammatory
], though may be harmful in some cases , 2) PKCθ signaling in T
Lymphocytes, involved in T-cell activation and NF-κB signaling [
], 3) iCOS-iCOSL signaling in
T-helper cells, resulting in activation and migration of lymphocytes and secretion of both
antiand pro-inflammatory cytokines , 4) NFAT regulation of the immune response, involved
in cytokine production in leukocytes [
], 5) TH1 signaling, resulting in pro-inflammatory
response, compared to the anti-inflammatory TH2 signaling [
] and 6) dendritic cell
maturation, which acts as a link between innate and adaptive immunity [
Aging increased and RLX decreased expression of a common set of genes, namely those
associated with the major histocompatibility complex (MHC) which is involved in immune
system signaling (Table 4). This was supported by MetaCore (Thomson Reuters) analysis that
showed activation of antigen presentation by MHC class I & II in aging and inhibition with
RLX treatment (data not shown).
Analysis of DEGs by IPA to predict which cellular functions would be altered in aging or by
RLX, showed that aged female rats exhibit increased gene expression involved in migration
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Fig 3. RLX inhibits multiple inflammatory pathways activated in aged females. (a) Pathway analysis of female rats
suggest that multiple inflammatory signaling pathways are activated (orange) in aging and (b) RLX treatment inhibited
(blue) many of these signaling pathways. Due to the substantial number of pathways altered in female aging (93
pathways) and RLX treatment (58 pathways), only those pathways whose activation or inhibition was predicted by IPA
(orange or blue colors, respectively) are shown.
Calcium-induced T Lymphocyte Apoptosis
iCOS-iCOSL Signaling in T Helper Cells
PKCθ Signaling in T Lymphocytes
Right column indicates which genes are common between aging and RLX groups which RLX reverses compared to the aging group. Arrows indicated direction of
change by RLX. FCGR3A/B: Encodes receptor for Fc portion of immunoglobin G. HLA: human leukocyte antigen; gene complex which forms the major
histocompatibility complex. LOC: indicates genes with uncertain functions.
PLOS ONE | https://doi.org/10.1371/journal.pone.0190935
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Gene reversal by relaxin in female rats
Activates Inhibits FCGR3A/FCGR3B(#), HLA-DQA1(#), HLA-DRA(#), HLA-DRB5(#), LOC1009009593/RT
HLA-DQA1(#), HLA-DRA(#), HLA-DRB5(#), LOC1009009593/RT(#)
Fig 4. DEG analysis in female LV to predict cellular functions. IPA shows that aging leads to extensive alterations of
gene expression resulting in leukocyte activation (panel a) and antigen presentation (panel b). RLX significantly
reduced the expression of multiple genes associated with immune activation (panel c) and antigen presentation (panel
d); that is, RLX largely reversed the effect of aging on the immune cell response. Orange centers denote activated
function. Blue centers denote inhibited function. Green genes indicate reduced expression. Red genes indicated
increases in expression. Orange, Blue and Yellow lines: the altered genes lead to activation, inhibition of function or is
acting in a way opposite to the current literature, respectively. Brown lines: the function of the gene is unclear in the
and activation of leukocytes and increased antigen presentation (Fig 4A and 4B), and these
functions are significantly reversed by RLX (Fig 4C and 4D), supporting RLXs role in MHC
In addition to the signaling pathways that are reversed by RLX based on IPA, MetaCore
analysis also showed that the classical complement cascade is significantly activated in aging
(Fig 5A) and reversed by RLX treatment (Fig 5B) in female rats.
IPA pathway analysis of male rats (Fig 6A and 6B) show a significant decrease in dendritic cell
maturation with age (Fig 6A) and inhibition of EIF2 and IL-6 signaling with RLX treatment
(Fig 6B). These data, again, support the notion that there are major differences in the
regulation of gene expression in male and female rats with aging and RLX treatment. MetaCore
analysis in male rats suggest that multiple pathways are inhibited by RLX: IL-1, CCR1, CCL2,
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Fig 5. Classical complement pathway activity in female LV in aging and aging plus RLX. The classical complement
cascade can result in an opsonization, or marking of cells for phagocytosis, and lysis of bacteria. It can also result in an
augmented inflammatory response. A. Effect of aging on complement component activation. B. Effect of RLX on
reversal of complement activity. Red boxes indicate significant alterations of specific complement components. Red
and blue thermometer markers next to specific components indicate increased and decreased component expression,
respectively. For key describing complement components, see https://portal.genego.com/help/MC_legend.pdf.
HMGB1/RAGE and LPS signaling, though these are not necessarily activated by aging (not
shown). IPA analysis in male rats, did not show an activation of immune cell recruitment in
aging, though RLX significantly repressed immune cell activity, recruitment and accumulation
These data and Tables 1±3 indicate that gender plays a vital role in terms of which genes are
altered in aging and disease, and that treatments can affect males and females in different
Based on higher IFNγ, IL-6, MHC gene expression, increased complement activation, and
prediction of increased inflammatory/immunce cell activity by IPA in aging female but not male
rats, we first investigated the effects of aging on macrophage and T-cell recruitment in female
ventricular tissue by immunofluorescence (Fig 7A±7C). Tissue macrophage infiltration
measured with the marker F4/80 [
], increased significantly in aging (Fig 7B) compared to the
young controls (Fig 7A) and was dramatically decreased with RLX treatment (Fig 7C). Next,
we tested the male LV for macrophage accumulation (Fig 7D±7F). Male rats had significantly
less macrophages that did the corresponding female rats in each group. Moreover, male aging
(Fig 7E) did not significantly increase macrophage accumulation compared to the young male
rats (Fig 7D), and RLX did not cause a significant change compared to young or aged male
controls (Fig 7F). Although RLX slightly increased and decreased T-cell accumulation in
females and males, respectively, the significance is unclear because T-cells were sparse (<6/
high power field) in all groups (data not shown).
Our data show that aging promotes an inflammatory response in females, but not males, and
RLX reverses these effects in females, and suppresses many inflammatory genes in males. One
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Fig 6. Relaxin suppress immune cell recruitment and accumulation, in male ventricles. Male rats had negligible
pathway alterations (a) with aging or (b) RLX treatment. Blue bars indicate pathway inhibition. IPA in males (c)
showed that quantity, recruitment and accumulation of various immune cells were inhibited (green) by RLX. In males,
aging and RLX treatment altered 24 and 46 pathways, respectively; hence only pathways whose activation or inhibition
was predicted by IPA (orange or blue colors, respectively) are shown here.
may reasonably conclude that RLX is acting at a genomic level to suppress inflammatory and
immune responses in male and female rat heats. Others studies have bemoaned that only 10%
of immunology studies consider sex as an important variable [
]; and the current findings
reenforce this concern by showing that females and males exhibit drastically different gene
expression patterns and signaling pathway regulation, not only in aging, but also by relaxin
Reductions in IL-6 have been associated with beneficial effects in rheumatoid arthritis,
though with increased LDL in the plasma. Here, we showed that aging increased IL-6
transcription in females, while also significantly lowering the LDL receptor necessary to reduce
LDL concentration in the plasma. However, RLX reduced IL-6 and increased LDLR,
suggesting more LDL receptors and a greater efficacy at removing excess LDL cholesterol from the
plasma. These results suggest RLX may prove to be a beneficial therapy for patients suffering
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Fig 7. Effects of aging and RLX on macrophage accumulation in female ventricles. A-C. Female. Aged female left ventricles had significant macrophage
accumulation (B. 21.47±0.86 macrophages/hpf) compared to young controls (A. 15.57±0.96) and RLX reversed this aged-dependent macrophage accumulation (C.
11.72±1.26). D-F. Male. Male left ventricles showed no change in macrophage accumulation in aging (E. 2.13±0.51) or with RLX treatment (4.88±0.84) compared to
young (0.65±0.27) male rats, and had less macrophages than females in each group. G. Quantification of macrophage accumulation. Blue: DAPI; Green: F4/80
macrophage marker; HPF: high power field. Data is reported as mean ± SEM. N.S.: Not significant. 60x magnification. Scale bar: 50 μm.
from rheumatoid arthritis or coronary artery disease. In addition, IL-6 has been associated
with type II diabetes development, and as such, RLX may prove to be a beneficial therapy in
diabetes management [
The classical complement cascades converge on C3 components, which are necessary for
C5 convertase formation and lead to inflammation (the anaphylatoxins C3a, C4a and C5a
components) or bacteria lysis (C5b) . Our data (Fig 5) shows that the C3a and C4a
complement components are indeed two pieces, upregulated by aging and reversed by RLX. The
anaphylatoxins can also increase activation of macrophages, resulting in increased release of
inflammatory cytokines and stimulate the release of proteolytic enzymes which contribute to
ongoing tissue damage and increase endothelial surface thrombogenicity. Moreover, clinical
trials have shown that inhibition of C3 convertase attenuates both leukocyte attraction and
mortality in cardiopulmonary bypass patients . Taken together, our data suggest that relaxin
could be a potent therapy to regulate aberrant complement activity.
Hofmann and Frantz  suggested that targeting specific cytokines in pre-clinical studies
and clinical trials have failed to produce beneficial results due to the redundant nature of the
immune system. Our study shows that RLX significantly alters multiple cytokines and
signaling pathways in both genders, which could lead to improved suppression of inflammatory
related pathophysiology that rely on these redundant immune effects.
Franceschi and Campisi [
] report that there are at least eight possible mechanisms for
ªinflammagingº: 1) persistent leukocyte production of reactive molecules, 2) cytokine
production from damaged non-immune cells, 3) alterations in anabolic signaling, (e.g. TNFα and
IL6 signaling) altering insulin production, 4) ªself-debrisº from cell injury that accumulates over
time, activating the inflammatory response, 5) reduced ability of the aged body to sequester
infiltrated bacteria or viruses, 6) cellular senescence, 7) release of cytokines from damaged
mitochondria and 8) dysregulation of the complement system. Our data supports reasons 1, 3
and 7 in that aging increases cytokine transcript production and RLX reduces cytokine
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transcript expression and accumulation of macrophages in LV tissue in females.
Needless-tosay, further studies are needed on all possible mechanisms. Inflammation has been implicated
in many aspects of cardiovascular disease including LV remodeling [
], hypertrophy ,
stroke , HF , and AF [
]. The suppression of inflammatory responses by RLX
warrants further investigation considering its possible beneficial actions towards a therapy for
such inflammatory related diseases as AF, diabetes, obesity and HF.
A limitation of this study is the lack of a general consensus for quantification and analysis
of RNA-seq data [
]. The cut-off for filtering by RPKM is arbitrary and varies greatly in the
literature, from 0.1 [
] to greater than 5 [
]. To determine which RPKM value to use for
gene filtering, we obtained a freely available RNA-seq dataset analyzed using TopHat software
], and compared results using our current analysis software. An RPKM 1 showed good
agreement among the results between the two software packages. Another limitation is that
RNA-seq was performed only on LV tissue, and it is unknown if atria or RV behave similarly.
Finally, the experiments cannot discriminate whether RLX is removing the inflammatory
stimulus resulting in downregulation of inflammatory markers, or is regulating transcription of the
inflammatory markers themselves. The long-term effects following a short-term
administration of RLX in clinical trials suggests a direct effect on transcription, and the broad reduction
in multiple genes suggests a common nexus of action. Evidence suggests that multiple
proinflammatory cytokines are controlled at the transcriptional level through histone
modifications or phosphorylation.[
] Our lab showed that RLX can regulate phosphorylation of
], hence future studies should look into RLX's ability to regulate histone modification
and phosphorylation of genes.
This project used the University of Pittsburgh HSCRF Health Sciences Sequencing Core at
Children's Hospital for RNA library preparation and sequencing services.
Conceptualization: Guy Salama.
Data curation: Brian Martin, Beth Ann Gabris-Weber, Rajiv Reddy, Ansuman
Formal analysis: Brian Martin, Guillermo Romero, Ansuman Chattopadhyay, Guy Salama.
Funding acquisition: Guy Salama.
Investigation: Guillermo Romero, Guy Salama.
Methodology: Beth Ann Gabris-Weber.
Supervision: Guillermo Romero, Guy Salama.
Visualization: Brian Martin, Guy Salama.
Writing ± original draft: Brian Martin.
Writing ± review & editing: Ansuman Chattopadhyay, Guy Salama.
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