Hypoxia induced changes of SePP1 expression in rat preadipocytes and its impact on vascular fibroblasts

Abstract

Human adipose tissues secret a lot of cytokines involved in physiological and pathological activities. Inflammation around blood vessels is positively related to the severity of atherosclerosis. This study was to investigate the impact of adipokine SePP1 on vascular fibroblasts (VF) under a hypoxia condition might provide new evidence and methods for treatment of atherosclerosis. The mRNA and protein expression of IL-6, MCP-1 and SePP1 were detected in preadipocytes under normoxic (21% O2) and hyperoxic (4% O2) conditions, and the impact of IL-6, MCP-1 and SePP1 on VF was investigated. The preadipocytes were cultured under normoxic and hypoxic conditions. Then, the cell growth, and the mRNA and protein expression of inflammatory cytokines (IL-6, MCP-1 and SePP1) were detected. The VF were cultured in the medium collected from preadipocytes maintained under hypoxic and normoxic conditions, and the phenotypes, migration and type I collagen protein of VF were determined. Results showed that under the hypoxic condition, the proliferation of preadipocytes increased significantly (P<0.05), and the mRNA and protein expression of IL-6 and MCP-1 elevated markedly (P<0.05). However, the SePP1 expression reduced dramatically (P<0.05). After co-culture with VF, the VF transformed into myofibroblasts, accompanied by increased migration and elevated type I collagen expression (P<0.05). Thus, hypoxia may accumulate visceral fat and induce inflammatory state of preadipocytes, with reduced SePP1 expression, which might be involved in the occurrence and development of atherosclerosis.

Keywords: Preadipocyte; SePP1; hypoxia; interleukin 6; monocyte chemoattractant protein-1; type I collagen; vascular fibroblasts.

Figure 1

Morphology of perirenal preadipocytes. A: After incubation for 3 days; B: After incubation for 5 days; C: After incubation for 7 days; D: After incubation for 9 days.

Figure 2

Oil red O staining of preadipocytes after induction.

Figure 3

Preadipocytes after incubation under normoxic and hypoxic conditions. A: Cells incubated under the normoxic condition for 1 day following passaging; B: Cells incubated under the hypoxic condition for 1 day following passaging; C: Cells incubated under the normoxic condition for 3 day following passaging; D: Cells incubated under the hypoxic condition for 3 day following passaging.

Figure 4

mRNA expression of IL-6, MCP-1 and SePP1 in preadipocytes after incubation under the hypoxic and normoxic conditions, *p<0.05 vs normoxic condition.

Figure 5

In vitro culture of VF (×40). A: Tissue adherent to wall; B: Fibroblasts just migrated from the tissue; C: Clusters of fibroblasts.

Figure 6

Immunofluorescence staining of VF after co-culture with preadipocytes (×100). A: VF maintained with preadipocytes for 6 days under the hypoxic condition; B: VF maintained with preadipocytes for 3 days under the hypoxic condition; C: VF maintained with preadipocytes for 6 days under the normoxic condition; D: VF maintained for 6 days in blank control group. 1: Cell morphology under light microscope; 2: Nuclear staining; 3: Immunofluorescent staining of a-actin; 4: Total imaging of immunofluorescent and nuclear staining.

Figure 7

VF migration after incubation under normoxic and hypoxic conditions (×200). A: VE maintained in the medium collected from preadipocytes which were maintained under the hypoxic condition; B: VE maintained in the medium collected from preadipocytes which were maintained under the normoxic condition; A1 and B1: Culture for 1 day; A3 and B3: Culture for 3 days; A5 and B5: Culture for 5 days.