Engineering bioactive nanoparticles to rejuvenate vascular progenitor cells

Abstract

Fetal exposure to gestational diabetes mellitus (GDM) predisposes children to future health complications including type-2 diabetes mellitus, hypertension, and cardiovascular disease. A key mechanism by which these complications occur is through stress-induced dysfunction of endothelial progenitor cells (EPCs), including endothelial colony-forming cells (ECFCs). Although several approaches have been previously explored to restore endothelial function, their widespread adoption remains tampered by systemic side effects of adjuvant drugs and unintended immune response of gene therapies. Here, we report a strategy to rejuvenate circulating vascular progenitor cells by conjugation of drug-loaded liposomal nanoparticles directly to the surface of GDM-exposed ECFCs (GDM-ECFCs). Bioactive nanoparticles can be robustly conjugated to the surface of ECFCs without altering cell viability and key progenitor phenotypes. Moreover, controlled delivery of therapeutic drugs to GDM-ECFCs is able to normalize transgelin (TAGLN) expression and improve cell migration, which is a critical key step in establishing functional vascular networks. More importantly, sustained pseudo-autocrine stimulation with bioactive nanoparticles is able to improve in vitro and in vivo vasculogenesis of GDM-ECFCs. Collectively, these findings highlight a simple, yet promising strategy to rejuvenate GDM-ECFCs and improve their therapeutic potential. Promising results from this study warrant future investigations on the prospect of the proposed strategy to improve dysfunctional vascular progenitor cells in the context of other chronic diseases, which has broad implications for addressing various cardiovascular complications, as well as advancing tissue repair and regenerative medicine.

Hyaluronic Acid Hydrogels with Phototunable Supramolecular Cross-Linking for Spatially Controlled Lymphatic Tube Formation

“…To further investigate the impact of viscoelasticity on lymphatic tube formation, qRT-PCR was performed to compare the gene expression of LEC characteristic markers on both elastic and viscoelastic hydrogels (Figure D). Since 6 h was determined to be the ideal endpoint for lymphatic CLS formation, 12 h was selected as the endpoint for gene expression analysis to ensure that the signaling cascade in response to elastic and viscoelastic hydrogels can be fully captured. , After culturing human LECs for 12 h, viscoelastic hydrogels showed increased expression of lymphatic vessel endothelial hyaluronan receptor-1 ( LYVE-1 ), podoplanin , and prospero homeobox 1 ( Prox1 ) compared to LECs cultured on elastic hydrogels; all three genes are key lymphatic markers. As a homologue to the CD44 glycoprotein, LYVE-1 is used by LECs to interact with HA. , Podoplanin is the only known endogenous ligand for C-type lectin-like receptor-2 (CLEC-2), which is important for blood and lymphatic separation during embryonic development. − The transcription factor Prox1 is the master regulator of lymphatic phenotypes and vasculatures. , Therefore, elevated expression of key lymphatic markers LYVE-1 , podoplanin , and Prox1 suggests that viscoelastic hydrogels preserve the LEC phenotype, an important factor in enabling lymphatic CLS formation.…”

“…Images were acquired at 4× using an inverted light microscope (ECHO Revolve, San Diego, CA). Images were analyzed using kinetic analysis of vasculogenesis (KAV), a custom plug-in for FIJI. , To visualize lymphatic tube formation, human LECs cultured on hydrogels were fixed after 6 h, and samples were incubated with conjugated F-actin primary antibody and counterstained with DAPI. To visualize F-actin distribution and focal adhesion kinase (FAK), phalloidin and FAK antibodies were used.…”

“…To analyze the effect of hydrogel dynamics on lymphatic phenotypes, LECs were seeded on elastic and viscoelastic hydrogels and cultured for 12 h in EGM MV2 media, supplemented with 100 ng mL –1 VEGF-C, 50 ng mL –1 bFGF, and 1.5 μM S1P. The 12 h time point was selected to ensure that the signaling cascade in response to VEGF-C and mechanical stimulation was captured. , Each biological replicate was created by pooling RNA from three individual wells to collect a sufficient amount of RNA. At least three biological replicates ( n = 3) were collected per condition and analyzed with real-time qRT-PCR with triplicate readings as previously described .…”

“…The 12 h time point was selected to ensure that the signaling cascade in response to VEGF-C and mechanical stimulation was captured. 46,58 Each biological replicate was created by pooling RNA from three individual wells to collect a sufficient amount of RNA. At least three biological replicates (n = 3) were collected per condition and analyzed with real-time qRT-PCR with triplicate readings as previously described.…”

“…Since 6 h was determined to be the ideal endpoint for lymphatic CLS formation, 12 h was selected as the endpoint for gene expression analysis to ensure that the signaling cascade in response to elastic and viscoelastic hydrogels can be fully captured. 46,58 After culturing human LECs for 12 h, viscoelastic hydrogels showed increased expression of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), podoplanin, and prospero homeobox 1 (Prox1) compared to LECs cultured on elastic hydrogels; all three genes are key lymphatic markers. As a homologue to the CD44 glycoprotein, LYVE-1 is used by LECs to interact with HA.…”