Circulating fibrocyte levels correlate with left ventricular mass in middle-aged healthy adults without hypertension

Abstract

Background

Fibrocytes, circulating bone-marrow derived cells that differentiate into fibroblasts and myofibroblasts, are a major source of hypertensive arterial fibrosis and correlate with left ventricular (LV) mass in subjects with hypertension. We tested whether circulating fibrocytes levels correlate with LV mass in middle-aged adults without hypertension.

Methods

We measured peripheral blood fibrocyte levels and their activated phenotypes in 13 middle-aged, non-hypertensive adults and performed cardiac magnetic resonance imaging to assess LV mass.

Results

There was a strong correlation between total fibrocyte levels (CD45 + Col1+) and LV mass index (r = 0.71, p = 0.006), as well as fibrocyte subsets expressing the chemokine markers CCR2 (r = 0.60, p = 0.032), CCR5 (r = 0.62, p = 0.029), CCR7 (r = 0.60, p = 0.034), co-expression of CXCR4 and CCR2 (r = 0.62, p = 0.029), α-SMA+ (r = 0.57, p = 0.044), CD133 (r = 0.59, p = 0.036), and pSTAT6 (r = 0.64, p = 0.032).

Conclusions

Circulating fibrocytes are associated with LV mass index in middle-aged, non-hypertensive adults and may be a harbinger for the development of hypertension.

1. Introduction

Nearly half of all adults in the U.S. have hypertension [1], a major modifiable risk factor for cardiovascular disease. The prevalence of hypertension increases with age and correlates with arterial stiffness and decreased left ventricular (LV) compliance due to increased collagen deposition in the walls of large arteries [2]. In subjects with hypertension, LV hypertrophy, clinically defined as an increase in LV mass, is associated with adverse outcomes and progression of hypertensive heart disease [3]. Fibrocytes are bone marrow-derived cells that circulate in the blood and home to injured tissues where they differentiate into fibroblasts and myofibroblasts and contribute to fibrogenesis [4].

We previously reported that the blood concentration of fibrocytes positively correlate with LV mass index in subjects with hypertension [5]. In animal models of hypertension, aortic stiffening precedes and promotes the onset of systemic hypertension [6] and circulating fibrocytes contribute to adventitial collagen deposition within the aorta and increase stiffness of the vessel wall [7]. The causal relationship between aortic stiffening and the development of hypertension also holds in humans: In a large group of middle-aged adults enrolled in the Framingham Heart Study Offspring cohort, aortic stiffness correlated with incident hypertension, underscoring the concept that vascular stiffness is a precursor of hypertension and not merely its consequence [8].

This literature suggests a causal series of events leading to the onset of hypertension: Aortic adventitial collagen deposition and stiffening, in part mediated by homing of fibrocytes, results in LV hypertrophy and, subsequently, clinically detectable hypertension. We therefore performed a pilot study to test the hypothesis that the concentration of circulating fibrocytes and their activated phenotypes correlated with LV mass as determined by cardiac magnetic resonance imaging (CMR) in middle-aged, non-hypertensive adults.

2. Methods

2.1. Study participants

Following approval by the Institutional Review Board, volunteers aged 50–65 years without a history of hypertension, tobacco or medication use, or medical comorbidities were enrolled. Subjects underwent systemic blood pressure measurement using a manual sphygmomanometer, venipuncture with peripheral blood sampling for circulating fibrocytes, and CMR with gadolinium.

2.2. Measurement of circulating fibrocytes

A 10 mL sample of heparinized venous blood was obtained for the measurement of circulating fibrocytes. Quantitative FACS analysis was used to determine the blood concentration of fibrocytes (defined as CD45 + Col1+ cells), activated subset of fibrocytes including those defined as staining for phosphorylated (p) STAT6 and alpha-smooth muscle actin (α-SMA) + fibrocytes (CD45 + α-SMA + Col1+), fibrocyte subsets that expressed the stem cell marker CD133, and fibrocytes expressing chemokine receptors (CXCR4, CCR2, CCR5, CCR7, and in combination) as previously described [5]. Antibodies were purchased from BD Biosciences (San Jose, California, USA), and R&D Systems (Minneapolis, MN, USA). All the antibodies were purchased conjugated except anti-Col1. Anti-Col1 or control antibody was conjugated to FITC using DyLight conjugation kits (Thermo Fisher Scientific, Waltham, MA), using the manufacturer's instructions (Supplemental Table). Cells were processed and read on a FACS Canto II flow cytometer and analyzed using BD Diva software.

2.3. Cardiac magnetic resonance imaging

After peripheral intravenous access was obtained and ECG leads for gating and monitoring were placed, the subject was placed in the magnet with a phased array surface coil overlying the chest. Scout imaging was performed to localize the heart. LV volumes and function were determined using steady state free precession cine imaging with the following sequence parameters - TR 2.7 ms, TE 1.3 ms, flip angle 73°, FOV 300-350 mm, and resolution 1.8 × 1.4 × 8.0 mm. The temporal resolution of the image acquisition depended on underlying heart rate. A stack of short axis images that were 8 mm thick with 2 mm gap covered the LV from apex to base. Three long-axis images were obtained (4-chamber, 2-chamber, and 3-chamber). Analyses were performed using Argus software (Siemens Healthcare, Princeton, NJ) on a Leonardo workstation (Siemens Healthcare, Erlangen, Germany). End-diastolic and end-systolic endocardial and epicardial cavity areas were semi-automatically planimetered for each short-axis slice excluding papillary muscles and trabeculae from the endocardial contours. Total LV mass and mass index (LV mass/body surface area), end-diastolic volume, end-systolic volume, stroke volume, and cardiac output was calculated. Late gadolinium enhanced images were obtained 10–15 min after injection of 0.15 mmol/kg using a phase sensitive inversion recovery pulse sequence with the following sequence parameters: TR 7.1 ms, TE 3.4 ms, flip angle 25°, FOV 300-340 mm, resolution 1.8 × 1.3 × 8 mm, inversion time adjusted to null normal myocardium. The images were visually analyzed for the presence of focal late gadolinium enhancement.

2.4. Statistical analysis

Data were analyzed on a Macintosh computer using PRISM statistical software (Version 10.2.3, GraphPad Software, Inc., Boston, MA, USA). Correlation coefficient (r) was calculated using Spearman (non-parametric) correlation. Probability values (two-sided) were considered statistically significant if they were <0.05.

3. Results

A total of 13 subjects (5 men and 8 women with mean age 58 +/− 6 years) were enrolled. Manual blood pressure measurement was <130/80 mmHg for all participants at the time of enrollment. None of the subjects had evidence of focal late gadolinium enhancement on CMR. We found a strong correlation between the concentration of total fibrocytes (CD45 + Col1+ cells) and LV mass index (r = 0.71, p = 0.006) (Fig. 1). We found similar correlation for fibrocyte subsets that expressed the stem cell marker CD133 (r = 0.59, p = 0.036), and those expressing the homing chemokine receptors CCR2 (r = 0.60, p = 0.032), CCR5 (r = 0.62, p = 0.029), CCR7 (r = 0.60, p = 0.034), and co-expression of CXCR4 and CCR2 (r = 0.62, p = 0.029). Finally, we also found a correlation with LV mass index of the activated subset of fibrocytes, defined as staining for pSTAT6 (signaling molecule downstream of TGF-beta receptor; r = 0.64, p = 0.032) and expression of α-SMA (a marker of myofibroblasts; r = 0.57, p = 0.044).

Fig. 1.

Correlation coefficient with 95 % confidence intervals of left ventricular (LV) mass index as assessed by CMR imaging with Total (CD45+ Col1+) circulating fibrocytes (Panel A), and fibrocyte subsets including CD45+ Col1+ α-SMA+ (Panel B), CD45+ Col1+ phosphorylated (p) STAT6+ (Panel C), CD45+ Col1+ CD133+ (Panel D), CD45+ Col1+ CCR2+ (Panel E), CD45+ Col1+ CXCR4+ CCR2+ (Panel F), CD45+ Col1+ CCR5+ (Panel G), and CD45+ Col1+ CCR7+ (Panel H).

4. Discussion

Fibrocytes are bone marrow-derived cells that home to inflamed and injured tissue and promote tissue fibrosis by depositing collagen. In animal models, elevation of blood fibrocytes temporally precedes detectable arterial wall fibrosis and the development of systemic hypertension [6]. Compared to traditional biomarkers that indirectly represent the pathophysiologic process, fibrocytes may mechanistically contribute to the development of hypertension and the associated abnormal myocardial mechanics. In our pilot study, circulating fibrocyte levels positively correlated with LV mass index in healthy middle-aged, non-hypertensive adults, suggesting they may be a harbinger for the development of hypertension. Since fibrocytes secrete pro-hypertrophic mediators including TNF-α, interleukin(IL)-1 beta, IL-6, and basic fibroblast growth factor [4], it is also possible that LV mass is a direct effect of fibrocytes on the heart irrespective of the aorta.

The term “inflammaging” defines a chronic, low-grade inflammatory state associated with aging, wherein fibrosis and damage of many organs, including the heart, is thought to occur [9]. In one human study, the proportion of circulating fibrocytes in elderly individuals were significantly higher compared to younger individuals, and the differentiation capacity of the circulating fibrocytes into myofibroblasts was also significantly higher in the elderly cohort [10]. Investigators studying the individual contributions of matrix-producing cells in an animal model of hypertension refuted the concept that fibrosis was solely due to activation of resident fibroblasts in the aorta, instead they found that more than half of the collagen-producing cells of the aorta were bone marrow-derived fibrocytes [7]. This pathophysiologic response may contribute to the higher prevalence of hypertension with older age.

We recognize several limitations in our study. First, it is a small, pilot study and the results are primarily hypothesis-generating in nature. Second, we do not have longitudinal follow-up on our subjects so we do not know if any subsequently developed clinical hypertension. Third, we do not have information on aortic stiffness. Lastly, while we found correlations between circulating fibrocyte levels and LV mass index, this does not imply causation.

In conclusion, our findings suggest that elevated circulating fibrocyte levels precede the development of hypertension. Compared to biomarkers that indirectly represent the pathophysiologic process, fibrocytes directly contribute to cardiac fibrosis and LV mass and the abnormal arterial and myocardial mechanics seen in hypertensive heart disease. Whether our results represent a normal physiologic response to aging or a harbinger for the development of hypertension is unknown. If longitudinal, observational studies confirm that elevated levels of circulating fibrocytes precede the development of hypertension, a simple blood test could predict this very common cardiac risk factor. Moreover, pharmacologic targeting of novel fibrocyte-specific pathways could be explored as a mechanism to prevent the development of hypertension or attenuate the severity of hypertensive heart disease.

The following is the supplementary data related to this article.

Funding

This work was funded by the Alpha Phi Foundation Heart to Heart Grant AWD14593 (Keeley), the American Heart Association Innovative Project Award 23IPA1051256 doi:10.58275/AHA.23IPA1051256.pc.gr.171964 (Keeley), and the National Institutes of Health grants R01AI135128 and R01HL169974 (Mehrad).

Disclosures

None.

Ethical statement

Investigational Review Board approval was obtained for this study.

CRediT authorship contribution statement

Daniel S. Feuer: Writing – original draft, Data curation. Borna Mehrad: Writing – review & editing, Software, Investigation, Formal analysis. Ellen C. Keeley: Writing – original draft, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

None.