A local proinflammatory signalling loop facilitates adverse age-associated arterial remodeling

Abstract

Background: The coincidence of vascular smooth muscle cells (VSMC) infiltration and collagen deposition within a diffusely thickened intima is a salient feature of central arterial wall inflammation that accompanies advancing age. However, the molecular mechanisms involved remain undefined.

Methodology/principal findings: Immunostaining and immunoblotting of rat aortae demonstrate that a triad of proinflammatory molecules, MCP-1, TGF-β1, and MMP-2 increases within the aortic wall with aging. Exposure of VSMC isolated from 8-mo-old rats (young) to MCP-1 effects, via CCR-2 signaling, both an increase in TGF-β1 activity, up to levels of untreated VSMC from 30-mo-old (old) rats, and a concurrent increase in MMP-2 activation. Furthermore, exposure of young VSMC to TGF-β1 increases levels of MCP-1, and MMP-2 activation, to levels of untreated VSMC from old rats. This autocatalytic signaling loop that enhances collagen production and invasiveness of VSMC is effectively suppressed by si-MCP-1, a CCR2 antagonist, or MMP-2 inhibition.

Conclusions/significance: Threshold levels of MCP-1, MMP-2, or TGF-β1 activity trigger a feed-forward signaling mechanism that is implicated in the initiation and progression of adverse age-associated arterial wall remodeling. Intervention that suppressed this signaling loop may potentially retard age-associated adverse arterial remodeling.

Figure 1. Age and MCP-1 effect on expression of TGF-β1.

A. Immunofluorescence staining in rat aortic walls. MCP-1 (red), TGF-β1 (green), merged (yellow). Original magnification: X400. B. TGF-β1 (green) and α-SMA (red) immunocytostaining (n = 3, X400). C. ELISA for activated TGF-β1 protein (left panel) and Western blots (right panel, inset) and average data (right panel). Early passage young and old VSMC were treated with or without MCP-1 (50 ng/ml) for 24 hours. *p<0.05, old vs. young control; #p<0.05, young with treatment of MCP-1 v.s. young control.

Figure 2. Age effects on subcellular distribution of TGF-β1.

A. Western blots of TGF-β1 subcellular fractions with aging (upper panels) and average data of subcellular TGF-⇓1 protein fractions lower panel). B. Representative Western blots of TGF-β1 subcellular fractions with aging and MCP-1 treatment. Similar results were obtained from at least 3 separate experiments. Early passage young and old VSMC were treated with or without MCP-1 (50 ng/ml) for 24 hours. *p<0.05, old vs. young.

Figure 3. siRNA MCP-1.

A. Photomicrograph of MCP-1 silenced VSMC. Inset showing enlarged image (X1000). B. qRT-PCR. *p<0.05, siRNA MCP-1 vs. scramble in 30 mo VSMC. C. Western blots of MCP-1 in 30 mo VSMC (n = 3). D. Immunofluoscence staining of MCP-1 (green), MMP-2 (red), and TGF-β1 (red) (X400) in 30 mo VSMC treated with scramble (upper panels) and MCP-1 (50 nm, lower panels) siRNA. E. Representative of Western blots for MCP-1 and TGF-β1 and PAGE gelatin zymograph. Similar results were obtained from at least 3 separate experiments. *p<0.05 vs. scramble control.

Figure 4. Profile of MMP-2, TGF-β1 and downstream signaling molecules with aging and MCP-1 intervention.

A. Gelatin zymograph. Early passage young VSMC were treated with or without MCP-1 for 24 hours. B. Gelatin zymograph (upper panel); and average data (lower panel). Early passage young and old VSMC were treated with or without MCP-1 (50 ng/ml) plus vCCI (100 ng/ml) for 24 hours. C. Gelatin zymograph (n = 3). Early passage young VSMC were treated with or without MCP-1 (50 ng/ml) plus neutralizing antibody against MMP-2 (2 µg/ml) for 24 hours. D. Representative Western blots of MCP-1 and collagen I after MCP-1 treatment (left panels) and average data (right panels). Early passage young VSMC were treated with or without MCP-1 (50 ng/ml) plus neutralizing antibody against MMP-2 (2 µg/ml) for 24 hours. *p<0.05, vs. young; #p<0.05, vs MCP-1 treatment alone.

Figure 5. VSMC Invasion.

A. VSMC treated with or without MCP-1 (50 ng/ml) plus vCCi (150 ng/ml) or GM 6001 (15 µM). *p<0.05, vs. young; #p<0.05, vs MCP-1 treatment alone. B. Old VSMC with or without si-MCP-1 (50 nM). *p<0.05 si-MCP-1 vs. scramble control.

Figure 6. Effects of TGF-β1 on young VSMC.

A. Representative Western blots of MCP-1 (upper panel) and β-actin (lower panel) and zymograph (middle panel). B. VSMC invasion. Early passage young and old VSMC were treated with or without TGF-β1 (1, 10, 100 ng/ml) for 24 hours. *p<0.05, vs. young; #p<0.05, TGF-β1 treatment vs young.

Figure 7. Simplified schematic diagram of a MCP-1/MMP-2/TGF-β1 loop signaling pathway.