Faculty Bibliography

Angiopoietin-like protein 3 (ANGPTL3) is a hepatically secreted protein and therapeutic target for reducing plasma triglyceride-rich lipoproteins and low-density lipoprotein (LDL) cholesterol. Although ANGPTL3 modulates the metabolism of circulating lipoproteins, its role in triglyceride-rich lipoprotein assembly and secretion remains unknown. CRISPR-associated protein 9 (CRISPR/Cas9) was used to target ANGPTL3 in HepG2 cells (ANGPTL3^-/-) whereupon we observed ∼50% reduction of apolipoprotein B100 (ApoB100) secretion, accompanied by an increase in ApoB100 early presecretory degradation via a predominantly lysosomal mechanism. Despite defective particle secretion in ANGPTL3^-/- cells, targeted lipidomic analysis did not reveal neutral lipid accumulation in ANGPTL3^-/- cells; rather ANGPTL3^-/- cells demonstrated decreased secretion of newly synthesized triglycerides and increased fatty acid oxidation. Furthermore, RNA sequencing demonstrated significantly altered expression of key lipid metabolism genes, including targets of peroxisome proliferator-activated receptor α, consistent with decreased lipid anabolism and increased lipid catabolism. In contrast, CRISPR/Cas9 LDL receptor (LDLR) deletion in ANGPTL3^-/- cells did not result in a secretion defect at baseline, but proteasomal inhibition strongly induced compensatory late presecretory degradation of ApoB100 and impaired its secretion. Additionally, these ANGPTL3^-/-;LDLR^-/- cells rescued the deficient LDL clearance of LDLR^-/- cells. In summary, ANGPTL3 deficiency in the presence of functional LDLR leads to the production of fewer lipoprotein particles due to early presecretory defects in particle assembly that are associated with adaptive changes in intrahepatic lipid metabolism. In contrast, when LDLR is absent, ANGPTL3 deficiency is associated with late presecretory regulation of ApoB100 degradation without impaired secretion. Our findings therefore suggest an unanticipated intrahepatic role for ANGPTL3, whose function varies with LDLR status.

The severity of aortic stenosis (AS) is associated with acquired von Willebrand syndrome (AVWS) and gastrointestinal bleeding, leading to anemia (Heyde's syndrome). We investigated how anemia is linked with AS and AVWS using the LA100 mouse model and patients with AS. Induction of anemia in LA100 mice increased transforming growth factor (TGF)-β1 activation, AVWS, and AS progression. Patients age >75 years with severe AS had higher plasma TGF-β1 levels and more severe anemia than AS patients age <75 years, and there was a correlation between TGF-β1 and anemia. These data are compatible with the hypothesis that the blood loss anemia of Heyde's syndrome contributes to AS progression via WSS-induced activation of platelet TGF-β1 and additional gastrointestinal bleeding via WSS-induced AVWS.

PURPOSE/OBJECTIVE:Autologous bone grafts demonstrate osteoconductive, osteoinductive, and osteogenic properties. Hand surgeons commonly augment surgical fixation with autografts to promote fracture healing. This study compared the intrinsic stem cell-like properties of 2 commonly used autograft sources in hand surgery: the iliac crest and distal radius. METHODS:A total of 9 subjects who received an iliac crest bone graft and distal radius bone graft harvest as a part of the standard care of distal radius malunion or nonunion correction or scaphoid nonunion open reduction and internal fixation were enrolled in the study. Cells were isolated by serial collagenase digestion and subjected to fibroblast colony-forming units, osteogenesis, and adipogenesis assays. The expression levels of genes involved in osteogenesis and adipogenesis were confirmed using quantitative polymerase chain reaction. RESULTS:The cells isolated from the iliac crest bone graft compared with those isolated from the distal radius bone graft demonstrated significantly higher mean fibroblast colony-forming unit efficiency; increased osteogenesis, as measured using alizarin red quantification; increased adipogenesis, as measured using oil red O quantification; and higher expression levels of genes involved in osteogenesis and adipogenesis under the respective differentiation conditions. CONCLUSIONS:The cells isolated from the iliac crest bone graft demonstrated a higher fibroblast colony-forming unit capacity and an increased capability to undergo both osteogenesis and adipogenesis. CLINICAL RELEVANCE/CONCLUSIONS:Limited evidence exists comparing the intrinsic stem cell-like properties of the iliac crest and distal radius despite the widespread use of each source in hand and wrist surgery. The information from this investigation may assist hand and wrist surgeons with the selection of a source of autograft.

BACKGROUND/UNASSIGNED:gene to an adult mammalian heart deficient in PKP2 can arrest disease progression and significantly prolong survival. METHODS/UNASSIGNED:Experiments were performed using a PKP2-cKO (cardiac-specific, tamoxifen-activated deletion of plakophilin-2). The potential therapeutic, adeno-associated virus vector of serotype rh.74 (AAVrh.74)-PKP2a (PKP2 variant A; RP-A601) is a recombinant AAVrh.74 gene therapy viral vector encoding the human PKP2a. AAVrh.74-PKP2a was delivered to adult mice by a single tail vein injection either before or after tamoxifen-activated PKP2-cKO. PKP2 expression was confirmed by molecular and histopathologic analyses. Cardiac function and disease progression were monitored by survival analyses, echocardiography, and electrocardiography. RESULTS/UNASSIGNED:Consistent with prior findings, loss of PKP2 expression caused 100% mortality within 50 days after tamoxifen injection. In contrast, AAVrh.74-PKP2a-mediated PKP2a expression resulted in 100% survival for >5 months (at study termination). Echocardiographic analysis revealed that AAVrh.74-PKP2a prevented right ventricle dilation, arrested left ventricle functional decline, and mitigated arrhythmia burden. Molecular and histological analyses showed AAVrh.74-PKP2a-mediated transgene mRNA and protein expression and appropriate PKP2 localization at the cardiomyocyte intercalated disc. Importantly, the therapeutic benefit was shown in mice receiving AAVrh.74-PKP2a after disease onset. CONCLUSIONS/UNASSIGNED:These preclinical data demonstrate the potential for AAVrh.74-PKP2a (RP-A601) as a therapeutic for PKP2-related arrhythmogenic right ventricular cardiomyopathy in both early and more advanced stages of the disease.

The cardiovascular system generates and responds to mechanical forces. The heartbeat pumps blood through a network of vascular tubes, which adjust their caliber in response to the hemodynamic environment. However, how endothelial cells in the developing vascular system integrate inputs from circulatory forces into signaling pathways to define vessel caliber is poorly understood. Using vertebrate embryos and in vitro-assembled microvascular networks of human endothelial cells as models, flow and genetic manipulations, and custom software, we reveal that Plexin-D1, an endothelial Semaphorin receptor critical for angiogenic guidance, employs its mechanosensing activity to serve as a crucial positive regulator of the Dorsal Aorta's (DA) caliber. We also uncover that the flow-responsive transcription factor KLF2 acts as a paramount mechanosensitive effector of Plexin-D1 that enlarges endothelial cells to widen the vessel. These findings illuminate the molecular and cellular mechanisms orchestrating the interplay between cardiovascular development and hemodynamic forces.

In animals, cells often move as collectives to shape organs, close wounds, or-in the case of disease-metastasize. To accomplish this, cells need to generate force to propel themselves forward. The motility of singly migrating cells is driven largely by an interplay between Rho GTPase signaling and the actin network. Whether cells migrating as collectives use the same machinery for motility is unclear. Using the zebrafish posterior lateral line primordium as a model for collective cell migration, we find that active RhoA and myosin II cluster on the basal sides of the primordium cells and are required for primordium motility. Positive and negative feedbacks cause RhoA and myosin II activities to pulse. These pulses of RhoA signaling stimulate actin polymerization at the tip of the protrusions and myosin-II-dependent actin flow and protrusion retraction at the base of the protrusions and deform the basement membrane underneath the migrating primordium. This suggests that RhoA-induced actin flow on the basal sides of the cells constitutes the motor that pulls the primordium forward, a scenario that likely underlies collective migration in other contexts.

Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.

UNLABELLED:nurse cells as a model, we find that Pol II forms long-lived, transcriptionally poised clusters distinct from liquid droplets, which contain unbound and paused Pol II. Depletion of the Integrator complex endonuclease module, but not its phosphatase module or Pol II pausing factors disperses these Pol II clusters. Consequently, histone transcription fails to reach peak levels during S-phase and aberrantly continues throughout the cell cycle. We propose that Pol II clustering is a regulatory step occurring near promoters that limits rapid gene activation to defined times. ONE SENTENCE SUMMARY/UNASSIGNED:histone locus as a model, we show that clustered RNA polymerase II is poised for synchronous activation.