Circulating vascular endothelial growth factor (VEGF) ligands and receptors are central regulators of vasculogenesis, angiogenesis, and lymphangiogenesis. In response to VEGF ligand binding, VEGF receptor tyrosine kinases initiate the chain of events that transduce extracellular signals into endothelial cell responses such as survival, proliferation, and migration. These events are controlled by intricate cellular processes that include the regulation of gene expression at multiple levels, interactions of numerous proteins, and intracellular trafficking of receptor-ligand complexes. Endocytic uptake and transport of macromolecular complexes through the endosome-lysosome system helps fine-tune endothelial cell responses to VEGF signals. Clathrin-dependent endocytosis remains the best understood means of macromolecular entry into cells, although the importance of non-clathrin-dependent pathways is increasingly recognized. Many of these endocytic events rely on adaptor proteins that coordinate internalization of activated cell-surface receptors. In the endothelium of both blood and lymphatic vessels, epsins 1 and 2 are functionally redundant adaptors involved in receptor endocytosis and intracellular sorting. These proteins are capable of binding both lipids and proteins and are important for promoting curvature of the plasma membrane as well as binding ubiquitinated cargo. Here, we discuss the role of epsin proteins and other endocytic adaptors in governing VEGF signaling in angiogenesis and lymphangiogenesis and discuss their therapeutic potential as molecular targets.

BACKGROUND: The importance of mitochondria in normal heart function are well recognized and recent studies have implicated changes in mitochondrial metabolism with some forms of heart disease. Previous studies demonstrated that knockdown of the mitochondrial ribosomal protein S5 (MRPS5) by small interfering RNA (siRNA) inhibits mitochondrial translation and thereby causes a mitonuclear protein imbalance. Therefore, we decided to examine the effects of MRPS5 loss and the role of these processes on cardiomyocyte proliferation.

METHODS: We deleted a single allele of MRPS5 in mice and used left anterior descending coronary artery ligation surgery to induce myocardial damage in these animals. We examined cardiomyocyte proliferation and cardiac regeneration both in vivo and in vitro. Doxycycline treatment was used to inhibit protein translation. Heart function in mice was assessed by echocardiography. Quantitative real-time polymerase chain reaction and RNA sequencing were used to assess changes in transcription and chromatin immunoprecipitation (ChIP) and BioChIP were used to assess chromatin effects. Protein levels were assessed by Western blotting and cell proliferation or death by histology and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Adeno-associated virus was used to overexpress genes. The luciferase reporter assay was used to assess promoter activity. Mitochondrial oxygen consumption rate, ATP levels, and reactive oxygen species were also analyzed.

RESULTS: We determined that deletion of a single allele of MRPS5 in mice results in elevated cardiomyocyte proliferation and cardiac regeneration; this observation correlates with improved cardiac function after induction of myocardial infarction. We identified ATF4 (activating transcription factor 4) as a key regulator of the mitochondrial stress response in cardiomyocytes from Mrps5+/- mice; furthermore, ATF4 regulates Knl1 (kinetochore scaffold 1) leading to an increase in cytokinesis during cardiomyocyte proliferation. The increased cardiomyocyte proliferation observed in Mrps5+/- mice was attenuated when one allele of Atf4 was deleted genetically (Mrps5+/-/Atf4+/-), resulting in the loss in the capacity for cardiac regeneration. Either MRPS5 inhibition (or as we also demonstrate, doxycycline treatment) activate a conserved regulatory mechanism that increases the proliferation of human induced pluripotent stem cell-derived cardiomyocytes.

CONCLUSIONS: These data highlight a critical role for MRPS5/ATF4 in cardiomyocytes and an exciting new avenue of study for therapies to treat myocardial injury.

BACKGROUND: Excess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, a family of endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In this study, we determined the role of Epsins in macrophage-mediated metabolic regulation. We then developed an innovative method to therapeutically target macrophage Epsins with specially designed S2P-conjugated lipid nanoparticles, which encapsulate small-interfering RNAs to suppress Epsins.

METHODS: We used single-cell RNA sequencing with our newly developed algorithm MEBOCOST (Metabolite-mediated Cell Communication Modeling by Single Cell Transcriptome) to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular, and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. We performed this study using myeloid-specific Epsin double knockout (LysM-DKO) mice and mice with a genetic reduction of ABCG1 (ATP-binding cassette subfamily G member 1; LysM-DKO-ABCG1fl/+). The nanoparticles targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis.

RESULTS: We revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing lesional macrophage-specific nanoparticle delivery of Epsin small-interfering RNAs, we showed that silencing of macrophage Epsins diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport. In a LysM-DKO-ABCG1fl/+ mouse model, enhanced cholesterol efflux and reverse transport due to Epsin deficiency was suppressed by the reduction of ABCG1.

CONCLUSIONS: Our findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis by reducing CD36-mediated lipid uptake and increasing ABCG1-mediated cholesterol efflux.

Cell identity genes are distinct from other genes with respect to the epigenetic mechanisms to activate their transcription, e.g. by super-enhancers and broad H3K4me3 domains. However, it remains unclear whether their post-transcriptional regulation is also unique. We performed a systematic analysis of transcriptome-wide RNA stability in nine cell types and found that unstable transcripts were enriched in cell identity-related pathways while stable transcripts were enriched in housekeeping pathways. Joint analyses of RNA stability and chromatin state revealed significant enrichment of super-enhancers and broad H3K4me3 domains at the gene loci of unstable transcripts. Intriguingly, the RNA m6A methyltransferase, METTL3, preferentially binds to chromatin at super-enhancers, broad H3K4me3 domains and their associated genes. METTL3 binding intensity is positively correlated with RNA m6A methylation and negatively correlated with RNA stability of cell identity genes, probably due to co-transcriptional m6A modifications promoting RNA decay. Nanopore direct RNA-sequencing showed that METTL3 knockdown has a stronger effect on RNA m6A and mRNA stability for cell identity genes. Our data suggest a run-and-brake model, where cell identity genes undergo both frequent transcription and fast RNA decay to achieve precise regulation of RNA expression.

Endothelialization of engineered vascular grafts for replacement of small-diameter coronary arteries remains a critical challenge. The ability for an acellular vascular graft to promote endothelial cell (EC) recruitment in the body would be very beneficial. This study investigated epsins as a target since they are involved in internalization of vascular endothelial growth factor receptor 2. Specifically, epsin-mimetic UPI peptides are delivered locally from vascular grafts to block epsin activity and promote endothelialization. The peptide delivery from fibrin coatings allowed for controlled loading and provided a significant improvement in EC attachment, migration, and growth in vitro. The peptides have even more important impacts after grafting into rat abdominal aortae. The peptides prevented graft thrombosis and failure that is observed with a fibrin coating alone. They also modulated the in vivo remodeling. The grafts are able to remodel without the formation of a thick fibrous capsule on the adventitia with the 100 µg mL-1 peptide-loaded condition, and this condition enabled the formation of a functional EC monolayer in the graft lumen after only 1 week. Overall, this study demonstrated that the local delivery of UPI peptides is a promising strategy to improve the performance of vascular grafts.

Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo. Here, we developed a fragment of C. difficile toxin B (TcdB^FBD), which recognizes and inhibits a subclass of FZDs, FZD1/2/7, and examined whether targeting this FZD subgroup may offer therapeutic benefits for treating breast cancer models in mice. Utilizing 2 basal-like and 1 luminal-like breast cancer models, we found that TcdB^FBD reduces tumor-initiating cells and attenuates growth of basal-like mammary tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Furthermore, FZD1/2/7–positive cells are enriched in chemotherapy-resistant cells in both basal-like and luminal mammary tumors treated with cisplatin, and TcdB^FBD synergizes strongly with cisplatin in inhibiting both tumor types. These data demonstrate the therapeutic value of narrow-spectrum Wnt signaling inhibitor in treating breast cancers.

Impaired lymphatic drainage and lymphedema are major morbidities whose mechanisms have remained obscure. To study lymphatic drainage and its impairment, we engineered a microfluidic culture model of lymphatic vessels draining interstitial fluid. This lymphatic drainage-on-chip revealed that inflammatory cytokines that are known to disrupt blood vessel junctions instead tightened lymphatic cell-cell junctions and impeded lymphatic drainage. This opposing response was further demonstrated when inhibition of rho-associated protein kinase (ROCK) was found to normalize fluid drainage under cytokine challenge by simultaneously loosening lymphatic junctions and tightening blood vessel junctions. Studies also revealed a previously undescribed shift in ROCK isoforms in lymphatic endothelial cells, wherein a ROCK2/junctional adhesion molecule-A (JAM-A) complex emerges that is responsible for the cytokine-induced lymphatic junction zippering. To validate these in vitro findings, we further demonstrated in a genetic mouse model that lymphatic-specific knockout of ROCK2 reversed lymphedema in vivo. These studies provide a unique platform to generate interstitial fluid pressure and measure the drainage of interstitial fluid into lymphatics and reveal a previously unappreciated ROCK2-mediated mechanism in regulating lymphatic drainage.

Background:

Cardiac valve disease is observed in 2.5% of the general population and 10% of the elderly people. Effective pharmacological treatments are currently not available, and patients with severe cardiac valve disease require surgery. PROX1 (prospero-related homeobox transcription factor 1) and FOXC2 (Forkhead box C2 transcription factor) are transcription factors that are required for the development of lymphatic and venous valves. We found that PROX1 and FOXC2 are expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 and FOXC2 regulate cardiac valve development and disease is not known.

Methods:

We used histology, electron microscopy, and echocardiography to investigate the structure and functioning of heart valves from Prox1^ΔVEC mice in which Prox1 was conditionally deleted from VECs. Isolated valve endothelial cells and valve interstitial cells were used to identify the molecular mechanisms in vitro, which were tested in vivo by RNAScope, additional mouse models, and pharmacological approaches. The significance of our findings was tested by evaluation of human samples of mitral valve prolapse and aortic valve insufficiency.

Results:

Histological analysis revealed that the aortic and mitral valves of Prox1^ΔVEC mice become progressively thick and myxomatous. Echocardiography revealed that the aortic valves of Prox1^ΔVEC mice are stenotic. FOXC2 was downregulated and PDGF-B (platelet-derived growth factor-B) was upregulated in the VECs of Prox1^ΔVEC mice. Conditional knockdown of FOXC2 and conditional overexpression of PDGF-B in VECs recapitulated the phenotype of Prox1^ΔVEC mice. PDGF-B was also increased in mice lacking FOXC2 and in human mitral valve prolapse and insufficient aortic valve samples. Pharmacological inhibition of PDGF-B signaling with imatinib partially ameliorated the valve defects of Prox1^ΔVEC mice.

Conclusions:

PROX1 antagonizes PDGF-B signaling partially via FOXC2 to maintain the extracellular matrix composition and prevent myxomatous degeneration of cardiac valves.

Alanyl-transfer RNA synthetase 2 (AARS2) is a nuclear encoded mitochondrial tRNA synthetase that is responsible for charging of tRNA-Ala with alanine during mitochondrial translation. Homozygous or compound heterozygous mutations in the Aars2 gene, including those affecting its splicing, are linked to infantile cardiomyopathy in humans. However, how Aars2 regulates heart development, and the underlying molecular mechanism of heart disease remains unknown. Here, we found that poly(rC) binding protein 1 (PCBP1) interacts with the Aars2 transcript to mediate its alternative splicing and is critical for the expression and function of Aars2. Cardiomyocyte-specific deletion of Pcbp1 in mice resulted in defects in heart development that are reminiscent of human congenital cardiac defects, including noncompaction cardiomyopathy and a disruption of the cardiomyocyte maturation trajectory. Loss of Pcbp1 led to an aberrant alternative splicing and a premature termination of Aars2 in cardiomyocytes. Additionally, Aars2 mutant mice with exon-16 skipping recapitulated heart developmental defects observed in Pcbp1 mutant mice. Mechanistically, we found dysregulated gene and protein expression of the oxidative phosphorylation pathway in both Pcbp1 and Aars2 mutant hearts; these date provide further evidence that the infantile hypertrophic cardiomyopathy associated with the disorder oxidative phosphorylation defect type 8 (COXPD8) is mediated by Aars2. Our study therefore identifies Pcbp1 and Aars2 as critical regulators of heart development and provides important molecular insights into the role of disruptions in metabolism on congenital heart defects.

Background: Endothelial-to-mesenchymal transition (EndMT) is a dynamic process in which endothelial cells acquire mesenchymal properties and in turn contribute to tissue remodeling and growth. Previously, we found EndMT associated with mitral valve adaptation after myocardial infarction. Furthermore, mitral valve endothelial cells collected at 6 months post-myocardial infarction expressed the pan-leukocyte marker CD45 and EndMT markers. Additionally, mitral valve endothelial cells induced to undergo EndMT with TGF (transforming growth factor)-β1 strongly coexpressed CD45 but not CD11b or CD14. Pharmacologic inhibition of the CD45 PTPase (protein tyrosine phosphatase) domain in mitral valve endothelial cells blocked TGFβ-induced EndMT. This prompted us to speculate that, downstream of TGFβ, CD45 induces EndMT.

Methods: We activated the endogenous CD45 promoter in human endothelial colony forming cells (ECFCs) using CRISPR (cluster regularly interspaced short palindromic repeats)/inactive Cas9 (CRISPR-associated protein 9) transcriptional activation. Bulk RNA sequencing was performed on control ECFCs and CD45-positive ECFCs to identify transcriptomic changes. Three functional assays-cellular migration, collagen gel contraction, and transendothelial electrical resistance-were conducted to assess mesenchymal properties in CD45-positive ECFCs.

Results: Activation of the endogenous CD45 promoter in ECFC and 3 additional sources of endothelial cells induced expression of several genes implicated in EndMT. In addition, CD45-positive ECFCs showed increased migration, a hallmark of EndMT, increased collagen gel contraction, a hallmark of mesenchymal cells, and decreased cell-cell barrier integrity, indicating reduced endothelial function.

Conclusions: CD45 is sufficient to incite an EndMT phenotype and acquisition of mesenchymal cell properties in normal human ECFCs. We speculate that CD45, through its C-terminal PTPase domain, initiates signaling events that drive EndMT.