Single cell transcriptomics reveals early photoreceptor states, cell-specific transcript isoforms, and cancer-predisposing features.

Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin. Here, we used deep full-length single-cell RNA-sequencing to distinguish post-mitotic cone and rod developmental states and cone-specific features that contribute to retinoblastomagenesis. The analyses revealed early post-mitotic cone- and rod-directed populations characterized by higher THRB or NRL regulon activities, an immature photoreceptor precursor population with concurrent cone and rod gene and regulon expression, and distinct early and late cone and rod maturation states distinguished by maturation-associated declines in RAX regulon activity. Unexpectedly, both L/M cone and rod precursors co-expressed NRL and THRB RNAs, yet they differentially expressed functionally antagonistic NRL isoforms and prematurely terminated THRB transcripts. Early L/M cone precursors exhibited successive expression of lncRNAs along with MYCN, which composed the seventh most L/M-cone-specific regulon, and SYK, which contributed to the early cone precursors' proliferative response to RB1 loss. These findings reveal previously unrecognized photoreceptor precursor states and a role for early cone-precursor-intrinsic SYK expression in retinoblastoma initiation.

Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids.

Fluorescent reporter pluripotent stem cell-derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of guanine nucleotide-binding protein subunit alpha transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP alleles robustly and exclusively labeled immature and mature cones. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of the morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 µm3 per day from day 126 to day 153. Immobilized GNAT2-EGFP cone reporter organoids provide a valuable tool for investigating human cone development and disease.

Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids.

Fluorescent reporter pluripotent stem cell (PSC) derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of Guanine Nucleotide-Binding Protein Subunit Alpha Transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP allele robustly and exclusively labeled both immature and mature cones starting at culture day 34. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 cubic microns per day from day 126 to day 153. Immobilized GNAT2-EGFP cone reporter organoids provide a valuable tool for investigating human cone development and disease.

Effect of Genetic and Dietary Perturbation of Glycine Metabolism on Atherosclerosis in Humans and Mice.

Objective: Epidemiological and genetic studies have reported inverse associations between circulating glycine levels and risk of coronary artery disease (CAD). However, these findings have not been consistently observed in all studies. We sought to evaluate the causal relationship between circulating glycine levels and atherosclerosis using large-scale genetic analyses in humans and dietary supplementation experiments in mice. Methods: Serum glycine levels were evaluated for association with prevalent and incident CAD in the UK Biobank. A multi-ancestry genome-wide association study (GWAS) meta-analysis was carried out to identify genetic determinants for circulating glycine levels, which were then used to evaluate the causal relationship between glycine and risk of CAD by Mendelian randomization (MR). A glycine feeding study was carried out with atherosclerosis-prone apolipoprotein E deficient (ApoE-/-) mice to determine the effects of increased circulating glycine levels on amino acid metabolism, metabolic traits, and aortic lesion formation. Results: Among 105,718 subjects from the UK Biobank, elevated serum glycine levels were associated with significantly reduced risk of prevalent CAD (Quintile 5 vs. Quintile 1 OR=0.76, 95% CI 0.67-0.87; P<0.0001) and incident CAD (Quintile 5 vs. Quintile 1 HR=0.70, 95% CI 0.65-0.77; P<0.0001) in models adjusted for age, sex, ethnicity, anti-hypertensive and lipid-lowering medications, blood pressure, kidney function, and diabetes. A meta-analysis of 13 GWAS datasets (total n=230,947) identified 61 loci for circulating glycine levels, of which 26 were novel. MR analyses provided modest evidence that genetically elevated glycine levels were causally associated with reduced systolic blood pressure and risk of type 2 diabetes, but did provide evidence for an association with risk of CAD. Furthermore, glycine-supplementation in ApoE-/- mice did not alter cardiometabolic traits, inflammatory biomarkers, or development of atherosclerotic lesions. Conclusions: Circulating glycine levels were inversely associated with risk of prevalent and incident CAD in a large population-based cohort. While substantially expanding the genetic architecture of circulating glycine levels, MR analyses and in vivo feeding studies in humans and mice, respectively, did not provide evidence that the clinical association of this amino acid with CAD represents a causal relationship, despite being associated with two correlated risk factors.

Local M-CSF (Macrophage Colony-Stimulating Factor) Expression Regulates Macrophage Proliferation and Apoptosis in Atherosclerosis.

OBJECTIVE: Previous studies have shown that deficiency of M-CSF (macrophage colony-stimulating factor; or CSF1 [colony stimulating factor 1]) dramatically reduces atherosclerosis in hyperlipidemic mice. We characterize the underlying mechanism and investigate the relevant sources of CSF1 in lesions. Approach and Results: We quantitatively assessed the effects of CSF1 deficiency on macrophage proliferation and apoptosis in atherosclerotic lesions. Staining of aortic lesions with markers of proliferation, Ki-67 and bromodeoxyuridine, revealed around 40% reduction in CSF1 heterozygous (Csf1+/-) as compared with WT (wild type; Csf1+/+) mice. Similarly, staining with a marker of apoptosis, activated caspase-3, revealed a 3-fold increase in apoptotic cells in Csf1+/- mice. Next, we determined the cellular sources of CSF1 contributing to lesion development. Cell-specific deletions of Csf1 in smooth muscle cells using SM22α-Cre (smooth muscle protein 22-alpha-Cre) reduced lesions by about 40%, and in endothelial cells, deletions with Cdh5-Cre (VE-cadherin-Cre) reduced lesions by about 30%. Macrophage-specific deletion with LysM-Cre (lysozyme M-Cre), on the other hand, did not significantly reduce lesions size. Transplantation of Csf1 null (Csf1-/-) mice bone marrow into Csf1+/+ mice reduced lesions by about 35%, suggesting that CSF1 from hematopoietic cells other than macrophages contributes to atherosclerosis. None of the cell-specific knockouts affected circulating CSF1 levels, and only the smooth muscle cell deletions had any effect on the percentage monocytes in the circulation. Also, Csf1+/- mice did not exhibit significant differences in Ly6Chigh/Ly6Clow monocytes as compared with Csf1+/+. CONCLUSIONS: CSF1 contributes to both macrophage proliferation and survival in lesions. Local CSF1 production by smooth muscle cell and endothelial cell rather than circulating CSF1 is the primary driver of macrophage expansion in atherosclerosis.

Rosuvastatin Prevents the Exacerbation of Atherosclerosis in Ligature-Induced Periodontal Disease Mouse Model.

Periodontitis is a local and systemic inflammatory condition and a risk factor of atherosclerosis, but no studies investigated the effect of a statin on atherogenesis affected by severe periodontitis. In this study, we investigated the effect of rosuvastatin (RSV) on atherogenesis in Apolipoprotein E-deficient mice receiving silk ligature placement around the maxillary second molars. Mice with the ligature placement developed severe periodontitis and vascular inflammation. RSV significantly inhibited the development of periodontitis and vascular inflammation and remarkably blocked the increased lipid deposition and the atherogenic gene expression in the arterial wall and aortic sinus induced by severe periodontitis. To understand the mechanistic effect of RSV on periodontitis-associated atherogenesis, we investigated the in vitro effect of RSV on various effect of TNF-α, a major proinflammatory cytokine for periodontitis and atherogenesis. We found that RSV notably inhibited the TNF-α-induced osteoclast formation, endothelial cell phenotypic changes, foam cell formation, and the expression of CD47 and other oncogenes in arterial smooth muscle cells. Taken together, our study indicates that RSV prevents the exacerbation of atherosclerosis induced periodontitis by inhibiting local, systemic and vascular inflammation, as well as the expression of CD47 from arterial smooth muscle cells in mice.