Sympathetic denervation and vascular smooth muscle cell phenotype: implications for vascular graft therapies

Introduction: Both atherosclerosis and vascular graft failure are characterized by the transdifferentiation of vascular smooth muscle cells (VSMC) from a healthy, contractile phenotype to a synthetic or osteogenic phenotype. This transdifferentiation is accompanied by over-proliferation of cells and deposition of pathogenic extracellular matrix proteins and calcium deposits. As a result, the vessel or graft mechanical properties shift and the lumen narrows, reducing blood flow. This problem has been recognized in the field for decades, but no devices or therapies developed to date successfully prevent this transdifferentiation. Healthy arteries are innervated by sympathetic nerves to regulate VSMC contractility and phenotype. This interaction has been largely overlooked as a target for vascular graft therapies, and it may represent a new tool for the regulation of VSMC phenotype. The goal of this project is to investigate the ways in which sympathetic innervation regulates vascular remodeling processes and VSMC phenotype. To study this, we have developed novel murine models of arterial denervation using the specific dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Our hypothesis is that sympathetic denervation of the murine femoral artery will lead to pathogenic transdifferentiation of arterial VSMC, while treatment of vascular cells with neuronal signaling factors may help to regulate contractile phenotypes.

Materials and Methods: The femoral arteries of BALB/c mice (both male and female) were treated with subcutaneous injection of 6-OHDA (1.0 mg/mL, 50 µL) or buffer vehicle solution weekly for 4 weeks. Blood flow to the skin of each hind paw before and after treatment was assessed by laser Doppler imaging. Femoral arteries and peri-adventitial tissue were harvested for histology and immunofluorescence at one (n=5) and four (n=6) weeks. Sympathetic nerve density was measured as the number of sympathetic nerve fibers (tyrosine hydroxylase-positive) normalized to the medial layer area based on immunofluorescence. Immunofluorescence for contractile proteins (smoothelin), macrophages (F4/80), and endothelial cells (CD31) was also performed. To probe signaling pathways between sympathetic nerves and VSMC, early passage synthetic human aortic smooth muscle cells (HAoSMC) were treated with sympathetic neurotransmitter norepinephrine (NE, alpha- and beta-adrenergic receptor ligand) and synthetic phenylephrine (PE, alpha-adrenergic receptor agonist) to determine changes in viability, morphology, and phenotype. Enzyme-linked immunosorbent assay (ELISA) and Western blot experiments are in progress to further characterize cell phenotype. Markers for contractile phenotypes (smoothelin, smooth muscle-myosin heavy chain, and organized α-smooth muscle actin), synthetic phenotypes (vimentin and proliferation marker Ki67), and osteogenic phenotypes (RUNX2, alkaline phosphatase, and osteocalcin) will all be assessed.

Results, Conclusions, and Discussions: Results & Discussion: Weekly injections of 6-OHDA result in sustained denervation (1.6 vs. 13.4 fibers/10,000 μm² [P=0.06, n=3]) in femoral arteries harvested at 4 weeks compared to control. There was reduced blood flow to the 6-OHDA-treated paw in male mice at low core temperatures [P< 0.01 compared to contralateral limb at 4 weeks, n=3]. Successful denervation and resulting blood flow changes can be seen in Figure 1. Expression of contractile marker smoothelin after 6-OHDA treatment is upregulated in females but there is no definite change in males based on preliminary analysis. All vessels demonstrate intact endothelial layers (CD31+) and macrophage presence (F4/80+).

Synthetic HAoSMC maintain high levels of viability at high concentrations (up to 1 mM) of norepinephrine and phenylephrine in vitro. Immunofluorescence staining suggests HAoSMC downregulate alpha 1A-adrenergic receptors in response to higher doses of norepinephrine or phenylephrine. This trend is consistent with literature and can be seen in Figure 2. VSMC express many adrenergic receptors, so expression of each subtype of alpha- and beta-adrenergic receptors will be analyzed to determine which receptor dominates the adrenergic response of VSMC. Phenotype characterization for these cells is ongoing to determine the effects of stimulating or blocking each of these receptors.

Conclusions: Femoral denervation with 6-OHDA leads to reduced blood flow to the limb at cold core temperatures. The mechanism and possible sex-dependence of these findings may be due to VSMC phenotype change, which is currently under active investigation. If a causal relationship can be identified between sympathetic denervation and VSMC pathology, engineering reinnervation could be a viable approach for future graft design.

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