Hemato-vascular specification requires arnt1 and arnt2 genes in zebrafish embryos

During embryonic development, a subset of cells in the mesoderm germ layer are specified as hemato-vascular progenitor cells, which then differentiate into endothelial cells and hematopoietic stem and progenitor cells. In zebrafish, the transcription factor npas4l, also known as cloche, is required for the specification of hemato-vascular progenitor cells. However, it is unclear if npas4l is the sole factor at the top of the hemato-vascular specification cascade. Here we show that arnt1 and arnt2 genes are required for hemato-vascular specification. We found that arnt1;arnt2 double homozygous mutant zebrafish embryos (herein called arnt1/2 mutants), but not arnt1 or arnt2 single mutants, lack blood cells and most vascular endothelial cells. arnt1/2 mutants have reduced or absent expression of etv2 and tal1, the earliest known endothelial and hematopoietic transcription factor genes. npas4l and arnt genes are PAS domain-containing bHLH transcription factors that function as dimers. We found that Npas4l binds both Arnt1 and Arnt2 proteins in vitro, consistent with the idea that PAS domain-containing bHLH transcription factors act in a multimeric complex to regulate gene expression. Our results demonstrate that npas4l, arnt1 and arnt2 act together as master regulators of endothelial and hematopoietic cell fate. Our results also demonstrate that arnt1 and arnt2 act redundantly in a transcriptional complex containing npas4l, but do not act redundantly when interacting with another PAS domain-containing bHLH transcription factor, the aryl hydrocarbon receptor. Altogether, our data enhance our understanding of hemato-vascular specification and the function of PAS domain-containing bHLH transcription factors.

Nitrate-Functionalized poly(ε-Caprolactone) Small-Diameter Vascular Grafts Enhance Vascular Regeneration via Sustained Release of Nitric Oxide

Artificial small-diameter vascular grafts (SDVG) fabricated from synthetic biodegradable polymers, such as poly(ε-caprolactone) (PCL), exhibit beneficial mechanical properties but are often faced with issues impacting their long-term graft success. Nitric oxide (NO) is an important physiological gasotransmitter with multiple roles in orchestrating vascular tissue function and regeneration. We fabricated a functional vascular graft by electrospinning of nitrate-functionalized poly(ε-caprolactone) that could release NO in a sustained manner via stepwise biotransformation in vivo. Nitrate-functionalized SDVG (PCL/NO) maintained patency following abdominal arterial replacement in rats. PCL/NO promoted cell infiltration at 3-months post-transplantation. In contrast, unmodified PCL SDVG showed slow cell in-growth and increased incidence of neointima formation. PCL/NO demonstrated improved endothelial cell (EC) alignment and luminal coverage, and more defined vascular smooth muscle cell (VSMC) layer, compared to unmodified PCL SDVG. In addition, release of NO stimulated Sca-1+ vascular progenitor cells (VPCs) to differentiate and contribute to rapid luminal endothelialization. Furthermore, PCL/NO inhibited the differentiation of VPCs into osteopontin-positive cells, thereby preventing vascular calcification. Overall, PCL/NO demonstrated enhanced cell ingrowth, EC monolayer formation and VSMC layer regeneration; whilst inhibiting calcified plaque formation. Our results suggested that PCL/NO could serve as promising candidates for improved and long-term success of SDVG implants.

Engineering Bioactive Nanoparticles to Rejuvenate Vascular Progenitor Cells

Fetal exposure to gestational diabetes mellitus (GDM) predisposes children to future health complications including hypertension and cardiovascular disease. A key mechanism by which these complications occur is through stress-induced dysfunction of vascular progenitor cells, including endothelial colony-forming cells (ECFCs). Although several approaches have been previously explored to restore endothelial dysfunction, their widespread adoption remains tampered by systemic side effects of adjuvant drugs and their limited efficacy. 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 vascular progenitor cells 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, which can be clinically-translated to address various cardiovascular complications, as well as toward a range of approaches in regenerative medicine.

Pathological Circulating Factors in Moyamoya Disease

Moyamoya disease (MMD) is a cerebrovascular disease that presents with vascular stenosis and a hazy network of collateral formations in angiography. However, the detailed pathogenic pathway remains unknown. Studies have indicated that in addition to variations in the of genetic factor RNF213, unusual circulating angiogenetic factors observed in patients with MMD may play a critical role in producing “Moyamoya vessels”. Circulating angiogenetic factors, such as growth factors, vascular progenitor cells, cytokines, inflammatory factors, and other circulating proteins, could promote intimal hyperplasia in vessels and excessive collateral formation with defect structures through endothelial hyperplasia, smooth muscle migration, and atypical neovascularization. This study summarizes the hypothesized pathophysiology of how these circulating factors affect MMD and the interactive modulation between them.

High cut-off dialysis mitigates pro-calcific effects of plasma on vascular progenitor cells

Mortality of patients with end-stage renal disease tremendously exceeds that of the general population due to excess cardiovascular morbidity. Large middle-sized molecules (LMM) including pro-inflammatory cytokines are major drivers of uremic cardiovascular toxicity and cannot be removed sufficiently by conventional high-flux (HFL) hemodialysis. We tested the ability of plasma from 19 hemodialysis patients participating in a trial comparing HFL with high cut-off (HCO) membranes facilitating removal of LMM to induce calcification in mesenchymal stromal cells (MSC) functioning as vascular progenitors. HCO dialysis favorably changed plasma composition resulting in reduced pro-calcific activity. LMM were removed more effectively by HCO dialysis including FGF23, a typical LMM we found to promote osteoblastic differentiation of MSC. Protein-bound uremic retention solutes with known cardiovascular toxicity but not LMM inhibited proliferation of MSC without direct toxicity in screening experiments. We could not attribute the effect of HCO dialysis on MSC calcification to distinct mediators. However, we found evidence of sustained reduced inflammation that might parallel other anti-calcifying mechanisms such as altered generation of extracellular vesicles. Our findings imply protection of MSC from dysfunctional differentiation by novel dialysis techniques targeted at removal of LMM. HCO dialysis might preserve their physiologic role in vascular regeneration and improve outcomes in dialysis patients.

Abstract 14044: Engineering Bioactive Nanoparticles to Rejuvenate Vascular Progenitor Cells

Introduction: Fetal exposure to gestational diabetes mellitus (GDM) predisposes children to future health complications including hypertension and cardiovascular disease. A key mechanism by which these complications occur is through stress-induced dysfunction of vascular progenitor cells, including endothelial colony-forming cells (ECFCs). In particular, overexpression of transgelin (TAGLN), also known as SM22α, in GDM-ECFCs is associated with actin cytoskeletal rearrangement, which results in reduced cell migration and impaired vasculogenesis. We hypothesized that bioactive nanoparticles (NPs) conjugated on the surface of GDM-ECFCs can provide a sustained pseudo-autocrine stimulation to improve in vitro and in vivo vasculogenesis. Methods & Results: We designed multilamellar lipid NPs with an average size of 147±63 nm in diameter to deliver small molecules SB-431542 (TGF-β inhibitor) directly to the surface of GDM-ECFCs. Bioactive NPs can be robustly conjugated to the surface of ECFCs using thiol-maleimide coupling without altering cell viability and key progenitor phenotypes. By controlling the release kinetic of TGF-β inhibitor from the NPs, we can normalize TAGLN expression and improve cell migration, a critical key step in establishing functional vascular networks. Moreover, bioactive NPs can restore the vasculogenic potential of GDM-ECFCs in both 2D Matrigel and 3D collagen assays. Finally, when transplanted into immunodeficient mice, GDM-ECFCs conjugated with bioactive NPs exhibit robust de novo blood vessel formation with high engraftment rate, comparable to normal ECFCs. Conclusions: Collectively, these findings highlight a simple, yet promising strategy to rejuvenate GDM-ECFCs and improve their therapeutic potentials, which can be clinically-translated to address various cardiovascular complications, as well as toward a range of approaches in tissue repair and regenerative medicine.

Abstract 16808: Divergent Mature Vascular Smooth Muscle Cell Fates Are Directed by Platelet-Derived Transcriptomic Codes in Aortic Aneurysms

Introduction: Loss of vascular smooth-muscle cells (vSMC) and subsequent defective replenishment of functional vSMC de novo are pathological hallmarks of abdominal aortic aneurysm (AAA). Evidence suggests that clusters of progenitors can be reprogrammed into vSMC lineage following vascular injury. However, the landscape of vSMC precursors and reprograming signatures that determine their fate in AAA is undefined. Hypothesis: We hypothesize that quiescent progenitor pools reside in aorta and undergo spatiotemporal fate decision into mature vSMC in AAA. We speculate that distinct ligand-receptor and transcriptional signals gear their pathogenic trajectories into dysfunctional vSMC in AAA. Methods & Results: Single-cell RNA-sequencing (scRNA-seq) identified vascular progenitor cells (Cd34 + Pdgfrα+) in murine (angiotensin II-induced) and human AAA. Pseudotime analysis tracked two differentiation fates of progenitors into divergent mature vSMC pools characterized by enrichment in contractile cytoskeleton machinery (Acta2 + Myh11 + ) or abnormally supplemented with matrix remodeling genes (Mmp2 + Col8a1 + ). Mmp2 + Col8a1 + clusters were dominant in AAA suggesting pathological reprograming in AAA. Gene regulatory network analysis mapped distinct libraries of upstream transcription factors in each mature vSMC states in AAA. Acta2 + Myh11 + and Mmp2 + Col8a1 + transcriptomes were driven by opposing actions of Pparγ and its repressor Runx1 respectively. Interestingly, cellular interaction analysis revealed that platelet-derived Pdgfβ fueled the phenotypic switch into Mmp2 + Col8a1 + vSMC via its receptor Pdgfrα. Dampening Pdgfrα signaling by systemic platelet depletion repressed Runx1 activation thereby increasing Pparγ transcriptomic activity in progenitors in the aortic wall. This rescued functional regeneration of vSMC (Acta2 + Myh11 + ) with contractile properties consistent with reduced aortic damage and diminished AAA incidence. Conclusions: Our data demonstrate that platelets are novel actors capable of awakening progenitor pools by coding their fate into pathogenic vSMC lineage in AAA. We provide evidence suggesting that strategies aimed at sustaining vSMC contractile destiny could encourage repair of vascular damage in AAA.