Thrombin increases inflammatory cytokine and angiogenic growth factor secretion in human adipose cells in vitro

Abstract

Background: Abdominal obesity is associated with pro-thrombotic and inflammatory states. Therefore, the purpose of this study was to examine the expression of thrombin receptors (PAR1 and PAR4) human adipose tissue and whether thrombin stimulates an inflammatory cytokine and growth factor profile in human adipose tissue.

Methods: Human adipose tissue, isolated preadipocytes and differentiated adipocytes were used in this study. PAR1 and PAR4 mRNA and protein were detected by RT-PCR and immunoblot analysis in both adipose tissue and adipose microvessels. In separate studies, IL-1beta, IL-6, MCP-1, TNF-alpha, IL-10, FGF-2, VEGF, and PDGF production were measured from adipose tissue (n = 5), adipocytes (n = 5), and preadipocytes (n = 3) supernatants with and without thrombin (1 or 10 U/ml; 24 hrs) treatment.

Results: Thrombin increased cytokine secretion of IL-1beta, IL-6, MCP-1 and TNF-alpha and growth factor secretion of VEGF from adipocytes along with MCP-1 and VEGF from preadipocytes. The direct thrombin inhibitor lepirudin given in conjunction with thrombin prevented the thrombin-mediated increase in cytokine and growth factor secretion.

Conclusion: Here we show that thrombin PAR1 and PAR4 receptors are present and that thrombin stimulates inflammatory cytokine generation and growth factor release in human adipose tissue and cells in vitro. These data suggest that thrombin may represent a molecular link between obesity and associated inflammation.

Figure 1

PAR1 and PAR4 are present in adipose tissue. A) PAR1 mRNA amplified from cDNA from adipose tissue from 5 individuals (lanes 1–5). The positive control (lane 6) contains cDNA from a human artery and the negative control (lane 7) contains PAR1 specific primers but no cDNA. B) PAR4 mRNA amplified from cDNA from adipose tissue from 5 individuals (lanes 1–5). The positive control (lane 6) contains cDNA from a human artery and the negative control (lane 7) contains PAR4 specific primers but no cDNA. C) Representative immunoblots of tissue or cells from 2 individuals (A and B). PAR1 and PAR4 protein detected in adipose tissue (lanes 2 and 4) and adipose microvessels (lanes 3 and 4) but only PAR4 is detected in Preadipocytes (lanes 7 and 9) and differentiated adipocytes (lanes 8 and 10). Lane 1 is a positive control lysate. Lane 6 contains the molecular weight marker. A total of 6 groups (whole tissue and isolated preadipocytes and differentiated adipocytes) were analyzed for PAR1 and PAR4.

Figure 2

Thrombin stimulates inflammatory cytokine secretion predominately from primary cultures of newly differentiated human adipocytes. Cultures of adipose tissue (n = 5), newly differentiated adipocytes (n = 5) or preadipocytes (n = 3) were stimulated with thrombin (1 U/ml) or thrombin (10 U/ml) in the presence or absence of a thrombin inhibitor (lepirudin) for 24 hours. PBS served as the negative control. The following cytokines were measured from the supernatants of confluent cultures. A) IL-1β; B) IL-6; C) TNF-α; D) MCP-1. Means ± SEM,. *P < 0.05 thrombin vs. PBS. ± P < 0.05 thrombin + lepirudin vs. thrombin.

Figure 3

Thrombin stimulates angiogenic growth hormone secretion predominately from primary cultures of newly differentiated human adipocytes. Cultures of adipose tissue (n = 5), newly differentiated adipocytes (n = 5) or preadipocytes (n = 3) were stimulated with thrombin (1 U/ml) or thrombin (10 U/ml) in the presence or absence of a thrombin inhibitor (lepirudin) for 24 hours. PBS served as the negative control. The following growth factors were measured from the supernatants of the cultures. A) FGF-2; B) PDGF and; C) VEGF. Means ± SEM *P < 0.05 thrombin vs. PBS. ± P < 0.05 thrombin + lepirudin vs. thrombin.