A link between smooth muscle cell death and extracellular matrix degradation during vascular atrophy

Abstract

Objective: High blood flow induces neointimal atrophy in polytetrafluoroethylene (PTFE) aortoiliac grafts and a tight external PTFE wrap of the iliac artery induces medial atrophy. In both nonhuman primate models, atrophy with loss of smooth muscle cells and extracellular matrix (ECM) begins at ≤4 days. We hypothesized that matrix loss would be linked to cell death, but the factors and mechanisms involved are not known. The purpose of this study was to determine commonly regulated genes in these two models, which we hypothesized would be a small set of genes that might be key regulators of vascular atrophy.

Methods: DNA microarray analysis (Sentrix Human Ref 8; Illumina, San Diego, Calif; ∼23,000 genes) was performed on arterial tissue from the wrap model (n = 9) and graft neointima from the graft model (n = 5) 1 day after wrapping or the switch to high flow, respectively. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was also performed. Expression of this vascular atrophy gene set was also studied after Fas ligand-induced cell death in cultured smooth muscle cells and organ cultured arteries.

Results: Microarray analysis showed 15 genes were regulated in the same direction in both atrophy models: 9 upregulated and 6 downregulated. Seven of nine upregulated genes were confirmed by qRT-PCR in both models. Upregulated genes included the ECM-degrading enzymes ADAMTS4, tissue plasminogen activator (PLAT), and hyaluronidase 2; possible growth regulatory factors, including chromosome 8 open reading frame 4 and leucine-rich repeat family containing 8; a differentiation regulatory factor (musculoskeletal embryonic nuclear protein 1); a dead cell removal factor (ficolin 3); and a prostaglandin transporter (solute carrier organic anion transporter family member 2A1). Five downregulated genes were confirmed but only in one or the other model. Of the seven upregulated genes, ADAMTS4, PLAT, hyaluronidase 2, solute carrier organic anion transporter family member 2A1, leucine-rich repeat family containing 8, and chromosome 8 open reading frame 4 were also upregulated in vitro in cultured smooth muscle cells or cultured iliac artery by treatment with FasL, which causes cell death. However, blockade of caspase activity with Z-VAD inhibited FasL-mediated cell death, but not gene induction.

Conclusion: Seven gene products were upregulated in two distinctly different in vivo nonhuman primate vascular atrophy models. Induction of cell death by FasL in vitro induced six of these genes, including the ECM-degrading factors ADAMTS4, hyaluronidase 2, and PLAT, suggesting a mechanism by which the program of tissue atrophy coordinately removes extracellular matrix as cells die. These genes may be key regulators of vascular atrophy.

Fig. 1

Venn diagram of significantly regulated genes during flow-mediated graft neointimal atrophy and wrap-mediated medial arterial atrophy. Fifteen genes were increased or decreased in both baboon models.

Fig. 2

RT-qPCR of genes found by microarray analysis to be regulated in the PTFE wrap and PTFE graft models of vascular atrophy. (A) ADAMTS4, see Kenagy et al{Kenagy, 2009 #12545} for graft data, (B) Ficolin 3 (FCN3), (C) Mustang 1 (MUSTN1), (D) Histone 2H2A (HIST2H2A), (E) tissue plasminogen activator (PLAT), (F) LRRC8, (G) C8orf4 or TC1, (H) SCLO2A1 or the prostaglandin transporter, PGT, (I) hyaluronidase 2 (HYAL2). Each point represents a triplicate determination of paired samples of RNA from an individual animal’s iliac arteries or PTFE graft intimas. Values are expressed as the ratio of wrapped/unwrapped or high blood flow/normal blood flow. The dotted line represents a ratio of 1 and the continuous line the mean value. *P<.02; ^#P=.063

Fig. 2

RT-qPCR of genes found by microarray analysis to be regulated in the PTFE wrap and PTFE graft models of vascular atrophy. (A) ADAMTS4, see Kenagy et al{Kenagy, 2009 #12545} for graft data, (B) Ficolin 3 (FCN3), (C) Mustang 1 (MUSTN1), (D) Histone 2H2A (HIST2H2A), (E) tissue plasminogen activator (PLAT), (F) LRRC8, (G) C8orf4 or TC1, (H) SCLO2A1 or the prostaglandin transporter, PGT, (I) hyaluronidase 2 (HYAL2). Each point represents a triplicate determination of paired samples of RNA from an individual animal’s iliac arteries or PTFE graft intimas. Values are expressed as the ratio of wrapped/unwrapped or high blood flow/normal blood flow. The dotted line represents a ratio of 1 and the continuous line the mean value. *P<.02; ^#P=.063

Fig. 3

The effect of FasL on expression of (A) tissue plasminogen activator (PLAT), (B) hyaluronidase2 (HYAL2), (C) LRRC8, (D) prostaglandin transporter (SLCO2A1), (E) C8orf4 (TC-1), (F) ficolin 3 (FCN3), and (G) Mustang 1 (MUSTN1) in cultured SMCs expressed as fold of 6 or 24 hour control. * P<.05 vs control; mean ± SEM of 3 experiments.

Fig. 4

The effect of FasL on expression of (A) tissue plasminogen activator (PLAT), (B) hyaluronidase2 (HYAL2), (C) LRRC8, (D) prostaglandin transporter (SLCO2A1), (E) C8orf4 (TC-1), (F) ficolin 3 (FCN3), and (G) Mustang 1 (MUSTN1) in arterial organ culture expressed as fold of control. * P<.05 vs control; ^† P=.06; mean ± SEM of 3–4 experiments.