Native extracellular matrix orientation determines multipotent vascular stem cell proliferation in response to cyclic uniaxial tensile strain and simulated stent indentation

Abstract

Cardiovascular disease is the leading cause of death worldwide, with multipotent vascular stem cells (MVSC) implicated in contributing to diseased vessels. MVSC are mechanosensitive cells which align perpendicular to cyclic uniaxial tensile strain. Within the blood vessel wall, collagen fibers constrain cells so that they are forced to align circumferentially, in the primary direction of tensile strain. In these experiments, MVSC were seeded onto the medial layer of decellularized porcine carotid arteries, then exposed to 10%, 1 Hz cyclic tensile strain for 10 days with the collagen fiber direction either parallel or perpendicular to the direction of strain. Cells aligned with the direction of the collagen fibers regardless of the orientation to strain. Cells aligned with the direction of strain showed an increased number of proliferative Ki67 positive cells, while those strained perpendicular to the direction of cell alignment showed no change in cell proliferation. A bioreactor system was designed to simulate the indentation of a single, wire stent strut. After 10 days of cyclic loading to 10% strain, MVSC showed regions of densely packed, highly proliferative cells. Therefore, MVSC may play a significant role in in-stent restenosis, and this proliferative response could potentially be controlled by controlling MVSC orientation relative to applied strain.

Keywords: Collagen; Cyclic tensile strain; ECM, Extracellular Matrix; Extracellular matrix; In-stent restenosis; MVSC, Multipotent vascular stem cell; Multipotent vascular stem cell; Proliferation; VSMC, Vascular smooth muscle cell.

Fig. 1

(A) Diagram showing how strips were cut from decellularized porcine carotid arteries in order to obtain strips with fibers predominantly oriented parallel or perpendicular to strain direction. (B) Strips pinned for cell seeding. (C) Setup of bioreactor chambers. Images of static indentation device (D) Before application of stent strut, (E) Top view with stent strut, (F) In 120 mL tube for addition of medium. Images of the dynamic indentation device (G) with strips loaded into the Bose bioreactor chamber, and (H) loaded into the Bose Biodynamic device with stent strut placed level with the top of the recellularized strips(I)Diagram showing 10% strain of tissue strips by stent strut application.

Fig. 2

The alignment distribution of rMVSC (A,B) nuclei (C, D) and f-actin (C, D) on decellularized porcine carotid artery samples left unstrained, or (A, C) strained parallel or (B, D) perpendicular to the direction of collagen fibers. (E, F, G, H) Representative images of rMVSC on decellularized porcine carotid artery samples (E, G) left unstrained or (F) strained parallel or (H) perpendicular to the direction of collagen fibers. n = 3.

Fig. 3

F-actin orientation of cells exposed to various loading regimes for 10 days. (A) Stent strut indentation decreases cell alignment in strain direction. Representative images of cells stained for f-actin for (B) unstrained and (C, E) statically and (D, F) dynamically loaded cells, both (C, D) near and (E, F) far from stent strut. n = 3 Scale bar = 100 μm *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 4

Cell number and proliferation. (A) Cell number for conditions with countable nuclei. (B) Percentage of Ki67+ nuclei for conditions with countable nuclei. (C) For conditions without countable nuclei, percentage of frames for which individual nuclei or Ki67+ nuclei cannot be counted. (D–F) Images of dynamically loaded samples, with arrows pointing to regions of high density, highly proliferative cells. n = 3 Scale bars = 400 μm.