Circulating VEGFB is a candidate biomarker for CVD risk and impacts monocytic inflammatory signalling processes via VEGFR1

C. Schulte (Hamburg)¹, J. D. Escobar (Hamburg)², C. Müller (Hamburg)³, T. Tong (Lübeck)⁴, K. Lackner (Mainz)⁵, A. Schulz (Mainz)⁶, S. Blankenberg (Hamburg)⁷, V. Salomaa (Helsinki (Finland))⁸, P. S. Wild (Mainz)⁹, T. Zeller (Lübeck)^10
1Universitäres Herz- und Gefäßzentrum Klinik für Kardiologie Hamburg, Deutschland; ²University Center of Cardiovascular Science, University Heart and Vascular Center Hamburg Department of Cardiology University Medical Center Hamburg, Hamburg Hamburg, Deutschland; ³Universitäres Herz- und Gefäßzentrum Allgemeine und Interventionelle Kardiologie Hamburg, Deutschland; ⁴Universität zu Lübeck Institut für Kardiogenetik Lübeck, Deutschland; ⁵Universitätsmedizin der Johannes Gutenberg-Universität Mainz Institut für Klinische Chemie und Labormedizin Mainz, Deutschland; ⁶Universitätsmedizin der Johannes Gutenberg-Universität Mainz Präventive Kardiologie und Präventive Medizin, Zentrum für Kardiologie Mainz, Deutschland; ⁷Universitäres Herz- und Gefäßzentrum Hamburg Klinik für Kardiologie Hamburg, Deutschland; ⁸Finnish Institute for Health and Welfare (THL) Helsinki (Finland), Finnland; ⁹Universitätsmedizin der Johannes Gutenberg-Universität Mainz Präventive Kardiologie und Medizinische Prävention Mainz, Deutschland; ¹⁰Universitätsklinikum Schleswig-Holstein Institut für Kardiogenetik Lübeck, Deutschland

Background: Monitoring monocytic gene expression patterns could aid to identify biomarkers for improved cardiovascular risk stratification.

Objective: To apply transcriptome screening analysis to identify novel biomarker candidates associated with obesity and diabetes as cardiovascular risk factors and test association with clinical endpoints. Next generation sequencing approaches may provide further insights into the molecular background of identified targets.

Methods: Transcriptome-wide monocytic gene expression changes were screened in relation to change in BMI over a time period of 5 years in a population-based cohort study including data from 1,092 participants. Serum VEGFB levels were quantified in a subset of the 1,092 subjects and validated in serum from n=1,895 individuals from an independent cohort study. In-vitro, THP-1 immoratlised monocytes were stimulated with recombinant VEGFB cells. Next generation sequencing was performed on the treated cells and the results were validated in THP-1 derived macrophages and primary monocytes. VEGF receptor 1 (VEGFR1) inhibition was performed on primary monocytes.

Results:  Decreased VEGFB mRNA levels strongly associated with increased BMI (p=2.8x10^-9) and increased mortality (HR_perSD=0.757, 95% CI=0.647-0.885, p=0.0005). Elevated circulating VEGFB levels significantly associated with increased risk for heart failure (HR_perSD=1.373, 95% CI=1.210-1.560, p=1.0×10^-6) and coronary artery disease (HR=1.018, 95% CI=1.003-1.034, p=0.019) in n=1,895 individuals from an independent cohort. In THP-1 culture, stimulation with recombinant VEGFB resulted in downregulation of VEGFB mRNA levels. Next generation sequencing returned an inflammation-associated pattern of dysregulated genes upon stimulation with VEGFB, including IL1B, TNF and NFKB – all of which are reported mediators of VEGFR1-receptor signalling. Additionally, next generation sequencing results revealed dysregulations in microRNAs, indicating an inflammation-related pattern (including miR-320a-3p and miR-let7a-5p). Inhibition of VEGF receptor 1 (VEGFR1) in primary human monocytes resulted in significant reduction of the inhibitory effect of recombinant VEGFB on VEGFB mRNA levels as well as IL1B and TNF expression but not NFKB expression. Washing recombinant VEGFB out of the cell culture medium reversed the inhibitory effect of recombinant VEGFB on cellular VEGFB mRNA levels.

Conclusion: Decreased monocytic gene expression of VEGFB was related to obesity, increased risk of T2DM  and all-cause mortality in  large general population. Circulating  VEGFB levels associated positively with BMI, diabetes, mortality as well as heart failure and coronary heart disease. Monocytes  seem to regulate VEGFB expression by a negative feed-back mechanism based on VEGFB protein. Our data suggests the underlying pathway to be mediated by VEGFR1 and inflammatory proteins IL1B and TNF.