Background
Hypertension is a significant health threat and an independent predictor of CV events including coronary heart disease, stroke, heart failure, and dementia in the general population [
1] and in RA [
2]. The prevalence of hypertension in rheumatoid arthritis (RA) is high, but varies between 4 and 72% producing inconsistent results in the repeated meta-analysis [
3‐
5]. Studies in RA population often indicated high rates of hypertension in patients with first stroke [
6‐
9] and the highest stroke risk in young patients < 50 years [
10]. Chronic inflammation, the hallmark of RA and an important contributor to the high CVD rate in these patients, is viewed as a major pathogenic process behind significant vascular dysfunction. Inflammation reduces artery elasticity and increases systemic vascular resistance, which consequently leads to an increased arterial blood pressure (BP) [
11‐
13]. Elevated C-reactive protein levels are associated with hypertension in the general population by reducing the production of endothelial nitric oxide and by triggering platelet activation and leukocyte adhesion. Pro-inflammatory cytokines TNF-a and IL6 maintain hypertension by inducing endothelial proliferation and increasing vascular permeability and blood volume [
14,
15].
Solid biological evidence connects insulin-like growth factor 1 (IGF1) with the regulation of endothelial cell function. Vascular endothelial and smooth muscle cells express IGF1 receptor (IGF1R), which mediates angio-protective effects of IGF1. Locally produced IGF1 supports proliferation and migration of endothelial progenitors essential for blood vessel reparation and controls oxidative stress triggered by inflammation [
16‐
18]. Circulating IGF1 is known to induce vasodilation, which contributes to the regulation of arterial BP and vascular tone, while deletion of IGF1 in mice enhanced mechanisms of vasoconstriction leading to hypertension. Clinically, both a direct and inverse relation between IGF1 levels and BP has been reported. Early studies with focus on patients with congenital or acquired abnormalities in GH/IGF1 production, indicated an association between high IGF1 production and hypertension [
19,
20]. Recent studies of general population with low prevalence of obvious endocrinological conditions reported an inverse association between IGF1 and BP [
21‐
24]. Low serum IGF1 is tightly linked to vascular changes in aging [
25] and obesity [
26] and to the increased cardiovascular morbidity and mortality [
27‐
30].
Disturbances in IGF1/IGF1R signaling are notable for RA pathology. It contributes to joint inflammation and affects homeostasis in chondrocytes, leukocytes and synovial fibroblasts [
31‐
33]. It has been shown that inflammation is directly related to high expression of IGF1R in leukocytes [
34], which might be responsible for higher pain perception in RA [
33,
35]. In a study combining two independent RA cohorts, we demonstrated that smoking and inflammation had additive suppressive effects on serum IGF1 levels [
36]. These reports imply IGF1/IGF1R signaling to be a natural protective response to inflammation and pain. Little is known whether IGF1 contributes to the prevalence of CVD in RA. Therefore, the objective of this study was to determine the importance of low IGF1 and alterations in IGF1/IGF1R signaling for CVD in RA and to assess whether similarities exist in patients without RA afflicted by ischemic stroke.
Methods
The RA cohort
This study involved 184 female RA patients consecutively enrolled at the Rheumatology Clinic of Sahlgrenska University Hospital, Gothenburg, and the Northern Älvsborg County Hospital, Uddevalla [
33,
36]. All the patients fulfilled the classification criteria for RA (ACR1987) and had median disease duration of 7 years. The dominating majority (89%) were RF and/or ACPA positive. Active anti-rheumatic treatment had 175 patients. Among those, monotherapy with MTX had 82 patients (47%), other DMARDs 3 patients (1.7%); combination therapy with DMARDS 45 patients (26%), TNF-a-inhibitors 30 patients (17%), and other biologics 15 patients (8.6%). The study is registered at the Clinical
Trials.gov with ID NCT03449589.
The ischemic stroke (IS) cohort
We use clinical and serological data collected at 3 months after index IS of 132 consecutive female patients recruited at the Sahlgrenska University Hospital in the frame of the Sahlgrenska Academy Study on Ischemic Stroke (SALHSIS) [
30,
37].
The studies are approved by the Swedish Ethical Review Authority (RA cohort, Dnr.659-11 and SALHSIS cohort, Dnr.Ö469-99) and are conducted in accordance with the ethical principles of the Helsinki Declaration and Good Clinical Practice. All patients provided written informed consent before enrolment into the study.
Cardiovascular risk
A 10-year risk of developing CVD was estimated (eCVR) using a digital version of the Framingham algorithm [
38] and included sex, age, systolic BP, treatment for hypertension, current smoking, diabetes mellitus, HDL, and TC.
Prospective follow-up
Five years after enrollment the RA patients were contacted for a structured telephone interview. The questions were asked for any CVD events, cases of type 2 diabetes (T2D) and current medication including anti-hypertensive drugs, anticoagulants, anti-diabetic drugs, and statins. The reported CVD events and changes in medications were then controlled against medical records and the Swedish National Patient Registry.
Hypertension was recorded if an incidental measure of systolic BP was > 140 mmHg, or diastolic BP was > 90 mmHg or pharmacological treatment for hypertension was used. The BP burden was calculated as a sum of systolic and diastolic BPs.
Disease activity score (DAS28) of RA was calculated based on assessment of 28 tender and swollen joints and ESR.
Blood sampling and storage
The samples were collected between 7 and 10 o’clock in the morning after overnight fasting. For serum preparation, the blood was obtained from the cubital vein into vacuum containers (BD Vacutainer) and for RNA preparation into PAXgene protection tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Serum samples were stored at − 70 °C and PAXtubes in − 20 °C until use.
Serological measurements
In RA samples, serum IGF1, total cholesterol (TC), triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured by photometry on Cobas 8000 (Roche Diagnostics, Switzerland). In the IS samples, serum IGF1 was measured with a radio-immune assay (Mediagnost, Reutlingen, Germany) [
39]. Plasma glucose levels were measured using FreeStyle Lite (Abbott Diabetes Care Ltd., Oxon, UK). Sandwich ELISAs were used to measure insulin (DY8056, R&D Systems, Minneapolis, MN, USA) and IL6 and IL1b (M9316 and M1934, respectively; Sanquin, Amsterdam, the Netherlands).
Gene expression analysis
Total mRNA was prepared using PAXgene Blood RNA kit (Qiagen). Complementary DNA was synthesized using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Amplification of the gene product was attained on a ViiA™7 Real-Time PCR (Applied Biosystems) using SYBR Green qPCR Mastermix (SA Biosciences, Qiagen) and primer pairs as reported [
33,
40]. Gene expression levels were calculated by the ddCt method and presented as relative quantity to the average expression in the IGF1
hi group.
Statistical analysis
The SPSS v.25 (IBMSPSS, Chicago, IL), GraphPad prism v.7,
www.open-epi.com, R v.3.3.0 (R Core Team, 2018) and R studio v.1.1.447 (
http://www.rstudio.com/) were used for the analysis. Data are presented as mean ± SD, median [IQR], or in absolute numbers. Missing data for BP (5%) and ESR (8%) were imputed using the linear regression (SPSS). The study cohorts were dichotomized into IGF1
hi and IGF1
low groups by the median level. Continuous data were analyzed using the Mann-Whitney
U test, the Kruskal-Wallis test followed by Dunn’s post hoc test, and Spearman’s correlation test. Relative risk prediction was done using the area under the receiver operative characteristic (ROC) curve. The Kaplan-Meier curves and the Mantel-Cox analysis were used to compare the groups. For the clustering analysis, the data were log normalized and ranked by row. Heatmaps and hierarchical clustering were performed in R, using the stats and gplots packages, Spearman correlation-based distances, and Ward2 linkage. All tests were two-tailed and conducted with 95% confidence.
Discussion
In this cross-sectional study, we explored longitudinal associations between serum IGF1 and early CVD in RA. We showed harmful consequences of normal-low levels of IGF1 for the increase in the eCVR in female RA patients < 50 years and premature development of new CVD events. The profile of new CVD events consisted of the established complications of hypertension including ischemic stroke, atrial fibrillation, and aorta aneurysm, which indicated that hypertension was an essential clinical sign of IGF1-related CVD of the studied patients. Indeed, the patients with low IGF1 demonstrated higher BP measures at baseline and a significant increase in treatment for hypertension at the prospective follow-up. These findings in RA patients are consolidated by the reference cohort of IS females of similar age, where a significant association between low IGF1, hypertension, and eCVR is also observed.
The cumulative knowledge gained in a recent meta-analysis supports a direct causality between low IGF1 and hypertension [
22]. Although the studied cohorts had no patients with extreme levels of IGF1, we observed a pronounced negative effect of normal-low IGF1 levels on the development of CVD and on its arterial localization. Our findings offer an appropriate molecular context to the high rate of stroke previously reported in young RA patients [
10]. We found that the IGF1-related increase in eCVR was most noticeable in young women < 50 years. This implies that the vascular effects related to IGF1 may precede severe atherosclerotic change in the vessel wall and predispose to its early development. Indeed, previous studies in RA connected early CV events with endothelial dysfunction [
11,
12]. Direct angioprotective properties of IGF1 for vascular endothelial cell function have been extensively studied and is experimentally verified [
16]. In clinical studies, substitution with IGF1 was often followed by resolution of hypertension and improvement of the intima-media thickness of the arterial vessel wall [
41‐
43]. Importantly, IGF1 has an immediate effect on repairing tissue damage after acute ischemic cerebral and myocardial injury [
39,
44,
45]. In RA, the factors enhancing IGF1 levels attracted less attention. In this study, we observed that the use of TNF-a inhibitors was associated with higher IGF1 levels, while patients with low IGF1 received mostly MTX monotherapy. Treatment with TNF-a inhibitors is suggested to improve hypertension through an endothelium-dependent mechanism [
46,
47], which could indicate a contribution of increasing IGF1 levels. We would expect that anti-rheumatic treatment assisting production and bioavailability of IGF1 to be an important tool to recover cardiovascular health in RA patients. Physical inactivity, a condition accumulated in long-lasting RA and in ischemic stroke [
8], is frequently associated with lower serum IGF1 [
48] that could be increased with physical exercise [
49]. Changing lifestyle to improve serum IGF1 levels could be an attractive alternative for RA patients [
50].
In the present study, we investigated how the difference in serum IGF1 changed the IGF1R signaling and its relation to eCVR. From analyzing leukocytes, we confirmed that serum IGF1 controlled the relative expression of proteins within the IGF1R pathway. Importantly, the relation between these proteins changes with hypertension, demonstrating its intimate connection with low IGF1. The unsupervised clustering of the IGF1-related proteins proposed the high CVR signature, which combined the low expression of IGF1, IRS1, and IRS2 with high serum IL6, insulin and plasma glucose and emphasized the functional role of serum IGF1 in the development of early CVD in RA. These findings are in line with our recent report in experimental arthritis, which puts forward the role of excessive inhibitory phosphorylation in IRS1 to create a condition of insulin/IGF1 resistance [
35]. The situation seems more complex in patients, where low IGF1 is concurrent with high IGF1R and low adaptors IRS1/2. Being an ancestor of pro-insulin, IGF1 modulates carbohydrate metabolism, which stimulates glucose transport and inhibits insulin sensitivity [
18]. In the general population and experimentally, IGF1 deficiency leads to glucose intolerance and T2D, the conditions rare among the studied RA patients. This was despite the fact that metabolic deviations as obesity, hyperlipidemia and hyperinsulinemia were expectedly frequent in IGF1
low group.
There are notable strengths and limitations in our study. Among strengths, this is to our knowledge the largest study of the relation between IGF1 levels and cardiovascular morbidity in RA, and the only one to explore the longitudinal association with the development of CVD events. An additional strength is the similarity of the RA and IS female cohorts, which comprise women of the same age. This study is done on the female cohort of RA patients and allows no extrapolation of the results on male RA patients. Additionally, the study is restricted to the risk of CV events and does not cover cerebrovascular health in female RA patients, since the vascular wall has not been evaluated. Hence, it is outlined among the limitations of our study. We believe that the chosen methodology does not affect the results obtained in the between-group comparison.
Acknowledgments
The authors appreciate the assistance of Drs. Mats Dehlin, Lovisa Leifsdottir, and Jan Bjersing, Rheumatology Clinics, Sahlgrenska University Hospital, and Dan Norberg, Rheumatology Unit, Uddevalla Hospital, for clinical evaluation of the patients.
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