Development and characterization of a fully humanized ACE2 mouse model.

Background: Many humanized angiotensin-converting enzyme 2 (ACE2) mouse models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection do not replicate human ACE2 protein expression and thus exhibit pathology infrequently observed in humans. To address this limitation, we designed and characterized a fully humanized ACE2 (hACE2) mouse by replacing all exons/introns of the mouse Ace2 locus with human DNA comprising the entire ACE2 gene and an upstream long noncoding RNA (LncRNA).

Results: Compared to the popular Keratin18 ACE2 (KRT18-ACE2, K18) mouse model of SARS-CoV-2 infection, hACE2 mice displayed a similar tissue expression profile of ACE2 as that seen in human tissues.

Activation of receptor-independent fluid-phase pinocytosis promotes foamy monocyte formation in atherosclerotic mice.

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of death worldwide. Clinical and experimental data demonstrated that circulating monocytes internalize plasma lipoproteins and become lipid-laden foamy cells in hypercholesterolemic subjects. This study was designed to identify the endocytic mechanisms responsible for foamy monocyte formation, perform functional and transcriptomic analysis of foamy and non-foamy monocytes relevant to ASCVD, and characterize specific monocyte subsets isolated from the circulation of normocholesterolemic controls and hypercholesterolemic patients.

Omicron XBB.1.5 subvariant causes severe pulmonary disease in K18-hACE-2 mice.

Owing to their continuous evolution, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) display disparate pathogenicity in mouse models. Omicron and its sublineages have been dominant worldwide. Compared to pre-Omicron VOCs, early Omicron subvariants reportedly cause attenuated disease in human ACE-2-expressing mice (K18-hACE-2).

Variant Increases the Risk of Developing VEGFR (Vascular Endothelial Growth Factor Receptor) Blocker-Induced Pulmonary Vascular Disease.

Background: G6PD (glucose-6-phosphate-dehydrogenase) is a key enzyme in the glycolytic pathway and has been implicated in the pathogenesis of cancer and pulmonary hypertension-associated vascular remodeling. Here, we investigated the role of an X-linked mutation (N126D polymorphism), which is known to increase the risk of cardiovascular disease in individuals from sub-Saharan Africa and many others with African ancestry, in the pathogenesis of pulmonary hypertension induced by a vascular endothelial cell growth factor receptor blocker used for treating cancer.

Methods And Results: CRISPR-Cas9 genome editing was used to generate the variant (N126D; ) in rats.

Prime editing in mice with an engineered pegRNA.

CRISPR editing involves double-strand breaks in DNA with attending insertions/deletions (indels) that may result in embryonic lethality in mice. The prime editing (PE) platform uses a prime editing guide RNA (pegRNA) and a Cas9 nickase fused to a modified reverse transcriptase to precisely introduce nucleotide substitutions or small indels without the unintended editing associated with DNA double-strand breaks. Recently, engineered pegRNAs (epegRNAs), with a 3'-extension that shields the primer-binding site of the pegRNA from nucleolytic attack, demonstrated superior activity over conventional pegRNAs in cultured cells.

Calponin 1 inhibits agonist-induced ERK activation and decreases calcium sensitization in vascular smooth muscle.

Smooth muscle cell (SMC) contraction and vascular tone are modulated by phosphorylation and multiple modifications of the thick filament, and thin filament regulation of SMC contraction has been reported to involve extracellular regulated kinase (ERK). Previous studies in ferrets suggest that the actin-binding protein, calponin 1 (CNN1), acts as a scaffold linking protein kinase C (PKC), Raf, MEK and ERK, promoting PKC-dependent ERK activation. To gain further insight into this function of CNN1 in ERK activation and the regulation of SMC contractility in mice, we generated a novel Calponin 1 knockout mouse (Cnn1 KO) by a single base substitution in an intronic CArG box that preferentially abolishes expression of CNN1 in vascular SMCs.

HDL regulates TGFß-receptor lipid raft partitioning, restoring contractile features of cholesterol-loaded vascular smooth muscle cells.

Background: Cholesterol-loading of mouse aortic vascular smooth muscle cells (mVSMCs) downregulates , a master regulator of the contractile state downstream of TGFβ signaling. this results in transitioning from a contractile mVSMC to a macrophage-like state. This process likely occurs based on studies in mouse and human atherosclerotic plaques.

is a Novel Long Noncoding RNA Promoting Vascular Smooth Muscle Inflammation via Scaffolding MKL1 and USP10.

Background: Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood.

Methods: Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA ().

CRISPR-Cas9 Long-Read Sequencing for Mapping Transgenes in the Mouse Genome.

Microinjected transgenes, both large and small, are known to insert randomly into the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. As the vast majority of transgenic mouse lines remain unmapped, we developed CRISPR-Cas9 Long-Read Sequencing (CRISPR-LRS) to ascertain transgene integration loci.

is a novel long noncoding RNA promoting vascular smooth muscle inflammation via scaffolding MKL1 and USP10.

Background: Activation of vascular smooth muscle cells (VSMCs) inflammation is vital to initiate vascular disease. However, the role of human-specific long noncoding RNAs (lncRNAs) in VSMC inflammation is poorly understood.

Methods: Bulk RNA-seq in differentiated human VSMCs revealed a novel human-specific lncRNA called IN flammatory M K L1 I nteracting L ong N oncoding RNA ( ).

PGC-1α senses the CBC of pre-mRNA to dictate the fate of promoter-proximally paused RNAPII.

PGC-1α is well established as a metazoan transcriptional coactivator of cellular adaptation in response to stress. However, the mechanisms by which PGC-1α activates gene transcription are incompletely understood. Here, we report that PGC-1α serves as a scaffold protein that physically and functionally connects the DNA-binding protein estrogen-related receptor α (ERRα), cap-binding protein 80 (CBP80), and Mediator to overcome promoter-proximal pausing of RNAPII and transcriptionally activate stress-response genes.

Generation and Comparative Analysis of an Mouse with Preferential Activity in Vascular Smooth Muscle Cells.

All current smooth muscle cell (SMC) mice similarly recombine floxed alleles in vascular and visceral SMCs. Here, we present an knock-in mouse and compare its activity with a mouse. Both drivers demonstrate equivalent recombination in vascular SMCs.

Mediterranean G6PD variant rats are protected from Angiotensin II-induced hypertension and kidney damage, but not from inflammation and arterial stiffness.

Rationale: Epidemiological studies suggest that individuals in the Mediterranean region with deficiency of glucose-6-phosphate dehydrogenase (G6PD) are less susceptible to cardiovascular diseases. However, our knowledge regarding the effects of G6PD deficiency on pathogenesis of vascular diseases caused by factors, like angiotensin II (Ang-II), which stimulate synthesis of inflammatory cytokines and vascular inflammation, is lacking. Furthermore, to-date the effect of G6PD deficiency on vascular health has been controversial and difficult to experimentally prove due to a lack of good animal model.

Of mice and human-specific long noncoding RNAs.

The number of human LncRNAs has now exceeded all known protein-coding genes. Most studies of human LncRNAs have been conducted in cell culture systems where various mechanisms of action have been worked out. On the other hand, efforts to elucidate the function of human LncRNAs in an in vivo setting have been limited.

Fate and State of Vascular Smooth Muscle Cells in Atherosclerosis.

Vascular smooth muscle cells (VSMCs) have long been associated with phenotypic modulation/plasticity or dedifferentiation. Innovative technologies in cell lineage tracing, single-cell RNA sequencing, and human genomics have been integrated to gain unprecedented insights into the molecular reprogramming of VSMCs to other cell phenotypes in experimental and clinical atherosclerosis. The current thinking is that an apparently small subset of contractile VSMCs undergoes a fate switch to transitional, multipotential cells that can adopt plaque-destabilizing (inflammation, ossification) or plaque-stabilizing (collagen matrix deposition) cell states.

Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.

Background: Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter.

Results: Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box.

MKL1 cooperates with p38MAPK to promote vascular senescence, inflammation, and abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy. Myocardin related transcription factor A (MRTFA, MKL1) is a multifaceted transcription factor, regulating diverse biological processes. However, a detailed understanding of the mechanistic role of MKL1 in AAA has yet to be elucidated.

CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean Variant.

Epidemiological studies suggest that individuals in the Mediterranean region with a loss-of-function, nonsynonymous single nucleotide polymorphism (S188F), in glucose-6-phosphate dehydrogenase () are less susceptible to vascular diseases. However, this association has not yet been experimentally proven. Here, we set out to determine whether the Mediterranean mutation confers protection from vascular diseases and to discover the underlying protective mechanism.

Coronary Disease-Associated Gene Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway.

Rationale: The gene encoding TCF21 (transcription factor 21) has been linked to coronary artery disease risk by human genome-wide association studies in multiple racial ethnic groups. In murine models, Tcf21 is required for phenotypic modulation of smooth muscle cells (SMCs) in atherosclerotic tissues and promotes a fibroblast phenotype in these cells. In humans, TCF21 expression inhibits risk for coronary artery disease.