Spatial control of spheroid assembly and phenotypic plasticity of vasa vasorum-associated pericytes on fibrous biomaterials

Introduction: The vasa vasorum (Latin: vessels of vessels) comprise an essential microvascular network that nourishes larger blood arteries and veins.1 The absence of a proper functioning vasa vasorum results in medial necrosis and vessel degeneration. Our prior work characterized pericytes associated with small arterial vasa vasorum as a rare population ( < 1%) within the adventitia of human ascending aorta and identified their curious ability to spontaneously assemble into 3D spheroids that sprout upon ex vivo culture on Matrigel substrates.2 Thus, we hypothesized that fibrous ECM matrix biophysical cues influence pericyte spheroid formation. Methods: To test the hypothesis, we cultured primary and immortalized human pericyte populations on nanofiber scaffolds of varying fiber diameters (200-800 nm) and architectures,3 growth factor reduced Matrigel, agarose-coated tissue culture plates, in hanging drops, and on standard tissue culture plastic as monolayers. Pericytes were harvested for qPCR, Western blotting, immunocytochemical and ELISA assays to evaluate their phenotypic profiles after 24 hr (monolayer, agarose-coated well, hanging drop, and Matrigel substrates) or up to 3 weeks in culture (nanofiber scaffolds). Spatial patterning of pericyte populations on nanofiber scaffolds was visualized using time lapse phase contrast microscopy. Results: Pericyte spheroid assembly was favored on 200 nm fibers while pericytes grew as monolayers on 800 nm fiber scaffolds (Fig 1A). Elongated structures formed from pericyte spheroid mergers on widely-spaced parallel fibers (Fig. 1B). Spatial control of pericyte spheroid assembly and monolayers was achieved using a combination of nanofiber architectures within the same scaffold (Fig. 1C). Pericyte spheroids exhibited up-regulation of the endothelial marker Pecam1 for Matrigel (40.9 fold, p=2.6e-5), agarose (27.7 fold, p=0.0028), hanging drop (38.0 fold, p=6.0e-6) and nanofiber scaffolds (1.49 fold, p=0.0964) when compared with monolayer cultured pericytes. Concurrently, a down-regulation in the smooth muscle cell/pericyte marker, calponin (Cnn) was observed for Matrigel (0.172 fold, p=6.1e-5), agarose (0.235 fold, p=9.1e-5) and hanging drop pericyte spheroids (0.204 fold, p=8.7e-5). Immunocytochemical detection revealed heterogenous expression of PECAM1/CD31 in hanging drop spheroids. ELISA detected an increase in VEGF secretion for pericyte hanging drop spheroids when compared with pericytes cultured on tissue culture plastic (554 ± 84 pg/ml vs 271 ± 79 pg/ml, p=0.039). Addition of the Rho kinase inhibitor Y27632 prevented and disrupted spheroid formation in hanging drops and nanofiber scaffolds, a finding that was reversible upon removal or degradation of the drug. Interestingly, when hanging drop spheroids were disrupted via trypsinization and re-plated on tissue culture plastic, cells down-regulated Pecam1/Cd31 (0.0275 fold, p=8.9e-4) and VegfA (0.118 fold, p=0.048) gene expression and lowered secretion of VEGF (1012 ± 33pg/ml vs 82 ± 17pg/ml, p=7.8e-9) to levels similar to pericytes trypsinized in parallel and re-plated on tissue culture plastic (60 ± 16 pg/ml vs 82 ± 17 pg/ml, p=0.370). Conclusions and Discussions: We demonstrated spatial control of vasa vasorum-associated pericyte spheroid assembly influenced by fiber matrix diameter and architecture and dependent on Rho kinase activity. Furthermore, this new knowledge that pericytes exhibit phenotypic plasticity to reversibly transdifferentiate toward the endothelial lineage may potentiate their application as spheroids for therapeutic control of post-natal angiogenesis of microvessels in diseased or engineered tissues.

Acknowledgements: This work was supported by NHBLI under award #R01HL131632 (JP), University of Pittsburgh Physicians (JP), UPMC Pellegrini Chair in Cardiothoracic Surgery (JP), the National Science Foundation Award # 1762468, #2107332, and #2119949 (ASN), the Institute of Critical Technologies and Science at Virginia Tech (ASN), and the Macromolecules Innovative Institute (ASN).

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